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Sample records for rad1 deletion enhances

  1. Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex.

    PubMed

    Tomkinson, A E; Bardwell, A J; Tappe, N; Ramos, W; Friedberg, E C

    1994-05-03

    The yeast recombination and repair proteins Rad1 and Rad10 associate with a 1:1 stoichiometry to form a stable complex with a relative molecular mass of 190 kDa. This complex, which has previously been shown to degrade single-stranded DNA endonucleolytically, also cleaves supercoiled duplex DNA molecules. In this reaction, supercoiled (form I) molecules are rapidly converted to nicked, relaxed (form II) molecules, presumably as a result of nicking at transient single-stranded regions in the supercoiled DNA. At high enzyme concentrations, there is a slow conversion of the form II molecules to linear (form III) molecules. The Rad1/Rad10 endonuclease does not preferentially cleave UV-irradiated DNA and has no detectable exonuclease activity. The nuclease activity of the Rad1/Rad10 complex is consistent with the predicted roles of the RAD1 and RAD10 genes of Saccharomyces cerevisiae in both the incision events of nucleotide excision repair and the removal of nonhomologous 3' single strands during intrachromosomal recombination between repeated sequences. In these pathways, the specificity and reactivity of the Rad1/Rad10 endonuclease will probably be modulated by further protein-protein interactions.

  2. Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.

    PubMed

    Yamamoto, Keisuke; Hara, Kiyotaka Y; Morita, Toshihiko; Nishimura, Akira; Sasaki, Daisuke; Ishii, Jun; Ogino, Chiaki; Kizaki, Noriyuki; Kondo, Akihiko

    2016-09-13

    Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common

  3. Rad1, rad10 and rad52 mutations reduce the increase of microhomology length during radiation-induced microhomology-mediated illegitimate recombination in saccharomyces cerevisiae.

    PubMed

    Chan, Cecilia Y; Schiestl, Robert H

    2009-08-01

    Abstract Illegitimate recombination can repair DNA double-strand breaks in one of two ways, either without sequence homology or by using a few base pairs of homology at the junctions. The second process is known as microhomology-mediated recombination. Previous studies showed that ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated recombination in trans during rejoining of unirradiated plasmids or during integration of plasmids into the genome. Here we show that radiation-induced microhomology-mediated recombination is reduced by deletion of RAD52, RAD1 and RAD10 but is not affected by deletion of RAD51 and RAD2. The rad52 mutant did not change the frequency of radiation-induced microhomology-mediated recombination but rather reduced the length of microhomology required to undergo repair during radiation-induced recombination. The rad1 and rad10 mutants exhibited a smaller increase in the frequency of radiation-induced microhomology-mediated recombination, and the radiation-induced integration junctions from these mutants did not show more than 4 bp of microhomology. These results suggest that Rad52 facilitates annealing of short homologous sequences during integration and that Rad1/Rad10 endonuclease mediates removal of the displaced 3' single-stranded DNA ends after base-pairing of microhomology sequences, when more than 4 bp of microhomology are used. Taken together, these results suggest that radiation-induced microhomology-mediated recombination is under the same genetic control as the single-strand annealing apparatus that requires the RAD52, RAD1 and RAD10 genes.

  4. Enhance, delete, incept: manipulating hippocampus-dependent memories.

    PubMed

    Spiers, Hugo J; Bendor, Daniel

    2014-06-01

    Here we provide a brief overview of recent research on memory manipulation. We focus primarily on memories for which the hippocampus is thought to be required due to its central importance in the study of memory. The repertoire of methods employed is expanding and includes optogenetics, transcranial stimulation, deep brain stimulation, cued reactivation during sleep and the use of pharmacological agents. In addition, the possible mechanisms underlying these memory changes have been investigated using techniques such as single unit recording and functional magnetic resonance imaging (fMRI). This article is part of a Special Issue entitled 'Memory enhancement'.

  5. Mec1/Tel1–dependent phosphorylation of Slx4 stimulates Rad1–Rad10 dependent cleavage of non–homologous DNA tails

    PubMed Central

    Toh, Geraldine W.-L.; Sugawara, Neal; Dong, Junchao; Toth, Rachel; Lee, Sang Eun; Haber, James E.; Rouse, John

    2015-01-01

    Budding yeast Slx4 interacts with the Rad1–Rad10 endonuclease that is involved in nucleotide excision repair (NER), homologous recombination (HR) and single–strand annealing (SSA). We previously showed that Slx4 is dispensable for NER but is essential for SSA. Slx4 is phosphorylated by the Mec1 and Tel1 kinases after DNA damage on at least six Ser/Thr residues, and mutation of all six residues to Ala reduces the efficiency of SSA. In this study, we further investigated the role of Slx4 phosphorylation in SSA, specifically in regulating cleavage of 3′ non–homologous (NH) DNA tails by Rad1-Rad10 during SSA and HR. Slx4 became phosphorylated after induction of a single double–strand break (DSB) during SSA and dephosphorylation coincided approximately with completion of repair. Slx4 is recruited to 3′ NH tails during DSB repair, but this does not require phosphorylation of Slx4. However, we identified specific damage-dependent Mec1/Tel1 site of Slx4 phosphorylation, Thr 113, that is required for efficient cleavage of NH tails by Rad1–Rad10. Consistent with these data, deletion of both Mec1 and Tel1 severely reduces the efficiency of NH DNA tail cleavage during HR. These data show that phosphorylation of Slx4 by Mec1 and Tel1 plays an important role in facilitating NH DNA tail cleavage during HR. PMID:20382573

  6. N-Terminal Deletion of Peptide:N-Glycanase Results in Enhanced Deglycosylation Activity

    PubMed Central

    Wang, Shengjun; Xin, Fengxue; Liu, Xiaoyue; Wang, Yuxiao; An, Zhenyi; Qi, Qingsheng; Wang, Peng George

    2009-01-01

    Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the β-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-ΔH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-ΔH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-ΔH1 towards native and non-native glycoproteins offers a potential biotechnological application. PMID:20016784

  7. A Codon Deletion at the Beginning of Green Fluorescent Protein Genes Enhances Protein Expression.

    PubMed

    Rodríguez-Mejía, José-Luis; Roldán-Salgado, Abigail; Osuna, Joel; Merino, Enrique; Gaytán, Paul

    2017-01-01

    Recombinant protein expression is one of the key issues in protein engineering and biotechnology. Among the different models for assessing protein production and structure-function studies, green fluorescent protein (GFP) is one of the preferred models because of its importance as a reporter in cellular and molecular studies. In this research we analyze the effect of codon deletions near the amino terminus of different GFP proteins on fluorescence. Our study includes Gly4 deletions in the enhanced GFP (EGFP), the red-shifted GFP and the red-shifted EGFP. The Gly4 deletion mutants and their corresponding wild-type counterparts were transcribed under the control of the T7 or Trc promoters and their expression patterns were analyzed. Different fluorescent outcomes were observed depending on the type of fluorescent gene versions. In silico analysis of the RNA secondary structures near the ribosome binding site revealed a direct relationship between their minimum free energy and GFP production. Integrative analysis of these results, including SDS-PAGE analysis, led us to conclude that the fluorescence improvement of cells expressing different versions of GFPs with Gly4 deleted is due to an enhancement of the accessibility of the ribosome binding site by reducing the stability of the RNA secondary structures at their mRNA leader regions. © 2016 S. Karger AG, Basel.

  8. Deletion Formation between the Two Salmonella Typhimurium Flagellin Genes Encoded on the Mini F Plasmid: Escherichia Coli Ssb Alleles Enhance Deletion Rates and Change Hot-Spot Preference for Deletion Endpoints

    PubMed Central

    Mukaihara, T.; Enomoto, M.

    1997-01-01

    Deletion formation between the 5'-mostly homologous sequences and between the 3'-homeologous sequences of the two Salmonella typhimurium flagellin genes was examined using plasmid-based deletion-detection systems in various Escherichia coli genetic backgrounds. Deletions in plasmid pLC103 occur between the 5' sequences, but not between the 3' sequences, in both RecA-independent and RecA-dependent ways. Because the former is predominant, deletion formation in a recA background depends on the length of homologous sequences between the two genes. Deletion rates were enhanced 30- to 50-fold by the mismatch repair defects, mutS, mutL and uvrD, and 250-fold by the ssb-3 allele, but the effect of the mismatch defects was canceled by the ΔrecA allele. Rates of the deletion between the 3' sequences in plasmid pLC107 were enhanced 17- to 130-fold by ssb alleles, but not by other alleles. For deletions in pLC107, 96% of the endpoints in the recA(+) background and 88% in ΔrecA were in the two hot spots of the 60- and 33-nucleotide (nt) homologous sequences, whereas in the ssb-3 background >50% of the endpoints were in four- to 14-nt direct repeats dispersed in the entire 3' sequences. The deletion formation between the homeologous sequences is RecA-independent but depends on the length of consecutive homologies. The mutant ssb allele lowers this dependency and results in the increase in deletion rates. Roles of mutant SSB are discussed with relation to misalignment in replication slippage. PMID:9055067

  9. Glandular epithelial AR inactivation enhances PTEN deletion-induced uterine pathology.

    PubMed

    Choi, Jaesung Peter; Zheng, Yu; Handelsman, David J; Simanainen, Ulla

    2016-05-01

    Phosphatase and tensin homolog (PTEN) deletion induces uterine pathology, whereas androgen actions via androgen receptor (AR) support uterine growth and therefore may modify uterine cancer risk. We hypothesized that the androgen actions mediated via uterine glandular epithelial AR could modify PTEN deletion-induced uterine pathology. To test our hypothesis, we developed uterine glandular epithelium-specific PTEN and/or AR knockout mouse models comparing the uterine pathology among wild-type (WT), glandular epithelium-specific AR inactivation (ugeARKO), PTEN deletion (ugePTENKO), and the combined PTEN and AR knockout (ugePTENARKO) female mice. The double knockout restricted to glandular epithelium showed that AR inactivation enhanced PTEN deletion-induced uterine pathology with development of intraepithelial neoplasia by 20 weeks of age. In ugePTENARKO, 6/10 (60%) developed intraepithelial neoplasia, whereas 3/10 (30%) developed only glandular hyperplasia in ugePTENKO uterus. No uterine pathology was observed in WT (n=8) and ugeARKO (n=7) uteri. Uterine weight was significantly (P=0.002) increased in ugePTENARKO (374±97 mg (mean±s.e.)) compared with WT (97±6 mg), ugeARKO (94±12 mg), and ugePTENKO (205±33 mg). Estrogen receptor alpha (ERα) and P-AKT expression was modified by uterine pathology but did not differ between ugePTENKO and ugePTENARKO, suggesting that its expressions are not directly affected by androgens. However, progesterone receptor (PR) expression was reduced in ugePTENARKO compared to ugePTENKO uterus, suggesting that PR expression could be regulated by glandular epithelial AR inactivation. In conclusion, glandular epithelial AR inactivation (with persistent stromal AR action) enhanced PTEN deletion-induced uterine pathology possibly by downregulating PR expression in the uterus. © 2016 Society for Endocrinology.

  10. Cyclosporin A markedly enhances superantigen-induced peripheral T cell deletion and inhibits anergy induction

    PubMed Central

    1992-01-01

    Cyclosporin A (CsA) is a well-known immunosuppressive agent that modulates immune tolerance in many ways. CsA can give rise to a state of long-term nonimmunosuppressed transplantation tolerance, but it can also aggravate autoimmune diseases, and provoke specific forms of autoimmunity. These effects, which are often paradoxical, remain largely unexplained. In this study, we investigated the effects of CsA on superantigen (superAg)-reactive peripheral T cells. The intravenous injection of either staphylococcal enterotoxin B (SEB), or Mls-1a cells into Mls-1b recipients, causes long-term in vitro nonresponsiveness (anergy) and partial elimination of the peripheral T cell receptor (TCR) V beta 8+/CD4+ and -V beta 6+/CD4+ T cell subsets, respectively. We report that CsA markedly enhances the peripheral elimination of SEB- and Mls-1a-reactive T cells such that up to 90% of the targeted CD4+/V beta subpopulations are deleted. The degree of deletion depends on the dose and the schedule of CsA administration, and the number of superAg injections. In situations where the extent of deletion is only moderate, we find that the remaining superAg-reactive T cells fail to develop anergy, unlike the T cells of control superAg-immunized mice. Higher doses of CsA are required to enhance T cell deletion (greater than or equal to 25 mg/kg/d, i.p.) than to impair anergy induction (greater than or equal to 6.25 mg/kg/d, i.p.). In view of these results, it appears that the degree of tolerance in CsA/superAg-treated mice depends on the balance between these opposing effects, i.e., enhancement of peripheral elimination versus the abrogation of anergy. The possibility of enhancing or preventing immune tolerance with a drug may have important clinical implications. PMID:1613464

  11. Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein

    SciTech Connect

    Arpino, James A. J.; Rizkallah, Pierre J.; Jones, D. Dafydd

    2014-08-01

    The beneficial engineered single-amino-acid deletion variants EGFP{sup D190Δ} and EGFP{sup A227Δ} have been studied. Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP{sup D190Δ} containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP{sup A227Δ} revealed that a ‘flipping’ mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.

  12. Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells.

    PubMed

    Moorthy, Sakthi D; Mitchell, Jennifer A

    2016-04-02

    Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus(129) x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

  13. Enhanced Klebsiella pneumoniae carbapenemase (KPC) expression from a novel Tn4401 deletion.

    PubMed

    Cheruvanky, Anita; Stoesser, Nicole; Sheppard, Anna E; Crook, Derrick W; Hoffman, Paul S; Weddle, Erin; Carroll, Joanne; Sifri, Costi D; Chai, Weidong; Barry, Katie; Ramakrishnan, Girija; Mathers, Amy J

    2017-04-03

    The Klebsiella pneumoniae carbapenemase gene (blaKPC) is typically located within the mobile transposon Tn4401 Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188bp deletion [lsqb]between istB and blaKPC[rsqb]) was present in 14% (39/281) clinical isolates. MICs for Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16, ≥16), ertapenem (≥8, 4) and cefepime (≥64, 4) than E. coli strains with Tn4401b (0.5, ≤0.5, ≤1). Quantitative RT-PCR demonstrated that Tn4401a had a 16-fold and Tn4401h a 4-fold increase in blaKPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants and showed that the Tn4401a and Tn4401h promoter sequences generated higher β-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. Activity of the isolated promoter P2 was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicate that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.

  14. ST2 deletion enhances innate and acquired immunity to murine mammary carcinoma.

    PubMed

    Jovanovic, Ivan; Radosavljevic, Gordana; Mitrovic, Maja; Juranic, Vanda Lisnic; McKenzie, Andrew N J; Arsenijevic, Nebojsa; Jonjic, Stipan; Lukic, Miodrag L

    2011-07-01

    ST2 is a member of the IL-1 receptor family and IL-33 was recently identified as its natural ligand. The IL-33/ST2 pathway regulates Th1/Th2 immune responses in autoimmune and inflammatory conditions, but the role of ST2 signaling in tumor growth and metastasis has not been investigated. We aimed to investigate whether ST2 gene deletion affects tumor appearance, growth, and metastasis, and antitumor immunity in an experimental metastatic breast cancer model. Deletion of ST2 in BALB/c mice bearing mammary carcinoma attenuated tumor growth and metastasis, which was accompanied by increased serum levels of IL-17, IFN-γ, and TNF-α and decreased IL-4. Tumor-bearing ST2-/- mice had significantly higher percentages of activated CD27high CD11bhigh NK cells, CD69+ and KLRG- NK cells and higher cytotoxic activity of splenocytes, NK cells, and CD8+ T cells in vitro. A significantly higher number of NK cells expressing IFN-γ were found in ST2-/- mice compared with WT recipients. In vivo depletion of CD8+ or NK cells revealed a key role for NK cells in enhanced antitumor immunity in ST2-/- mice. We report for the first time that suppressed breast cancer progression and metastasis in mice lacking ST2 corresponds mainly with enhanced cytotoxic activity of NK cells, and increased systemic Th1/Th17 cytokines. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Genomic deletion of a long-range bone enhancer misregulatessclerostin in Van Buchem disease

    SciTech Connect

    Loots, Gabriela G.; Kneissel, Michaela; Keller, Hansjoerg; Baptist, Myma; Chang, Jessie; Collette, Nicole M.; Ovcharenko, Dmitriy; Plajzer-Frick, Ingrid; Rubin, Edward M.

    2005-04-15

    Mutations in distant regulatory elements can negatively impact human development and health, yet due to the difficulty of detecting these critical sequences we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem disease (VB), a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis we characterized the expression of human sclerostin (sost) from normal (hSOSTwt) or Van Buchem(hSOSTvb D) alleles. Only the hSOSTwt allele faithfully expressed high levels of human sost in the adult bone and impacted bone metabolism, consistent with the model that the VB noncoding deletion removes a sost specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays we were able to identify a candidate enhancer element that drives human sost expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the E14.5 mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes.

  16. Deletion of a HoxD enhancer induces transcriptional heterochrony leading to transposition of the sacrum.

    PubMed Central

    Zákány, J; Gérard, M; Favier, B; Duboule, D

    1997-01-01

    A phylogenetically conserved transcriptional enhancer necessary for the activation of Hoxd-11 was deleted from the HoxD complex of mice by targeted mutagenesis. While genetic and expression analyses demonstrated the role of this regulatory element in the activation of Hoxd-11 during early somitogenesis, the function of this gene in developing limbs and the urogenital system was not affected, suggesting that Hox transcriptional controls are different in different axial structures. In the trunk of mutant embryos, transcriptional activation of Hoxd-11 and Hoxd-10 was severely delayed, but subsequently resumed with appropriate spatial distributions. The resulting caudal transposition of the sacrum indicates that proper vertebral specification requires a precise temporal control of Hox gene expression, in addition to spatial regulation. A slight time delay in expression (transcriptional heterochrony) cannot be compensated for at a later developmental stage, eventually leading to morphological alterations. PMID:9250683

  17. The effect of ionizing radiation on mRNA levels of the DNA damage response genes rad9, rad1 and hus1 in various mouse tissues.

    PubMed

    Zhang, Zhenya; Cai, Zeyuan; Li, Kaiming; Fang, Yu; An, Lili; Hu, Zhishang; Wang, Shihua; Hang, Haiying

    2015-01-01

    Rad9, Rad1 and Hus1 are essential genes conserved from yeast to humans. They form a heterotrimer complex (9-1-1 complex) that participates in the cell cycle checkpoint activation and DNA damage repair in eukaryotic cells. Rad9, Rad1 and Hus1 deficient cells are hypersensitive to ionizing radiation and mouse cells deleted for anyone of the three genes are highly sensitive to the killing by gamma rays. We propose that ionizing radiation-induced transcription of these genes is a mechanism by which cells respond to radiation-induced damage. In this study we used quantitative real-time RT-PCR(qPCR) to analyze the mRNA levels of Rad9, Rad1 and Hus1 in various tissues isolated from mice that were either mock irradiated or exposed to 10 Gy gamma radiation. Our results indicated that the mRNA levels of Rad9, Rad1 and Hus1 genes were very different among these tissues, and we found high natural levels of mRNA in the spleen, lung, ovary and testis of mice before exposure to radiation. The mRNA levels of the three genes were well correlated across these tissues, being high, medium or low in each of the tissues simultaneously. The mRNA levels of the three genes were analyzed at 2, 6, 12, 24 and 48 h after irradiation. In most tissues Rad9 was strongly induced at 2 and 12 h time points and Hus1 was strongly induced at 2, 12 and 48 h time points, but Rad1 was minimally induced in most of the tissues with the exception of slightly higher levels in the heart and lung tissues at the 48 h time point. These results suggest that the regulation mechanisms for the mRNA levels of the three genes in response to ionizing radiation are complex and not well orchestrated. We also detected the induction of Rad9 and Hus1 proteins in the heart and liver of the animals after irradiation, and found that Rad9 protein levels were highly induced in both the heart and liver, while the Hus1 protein levels were significantly induced only in the liver, suggesting that Rad9 and Hus1 protein levels are not

  18. Enhanced production of recombinant proteins with Corynebacterium glutamicum by deletion of insertion sequences (IS elements).

    PubMed

    Choi, Jae Woong; Yim, Sung Sun; Kim, Min Jeong; Jeong, Ki Jun

    2015-12-29

    In most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain. From the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA). Our findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C

  19. MtgA Deletion-Triggered Cell Enlargement of Escherichia coli for Enhanced Intracellular Polyester Accumulation

    PubMed Central

    Kadoya, Ryosuke; Matsumoto, Ken’ichiro; Ooi, Toshihiko; Taguchi, Seiichi

    2015-01-01

    Bacterial polyester polyhydroxyalkanoates (PHAs) have been produced in engineered Escherichia coli, which turned into an efficient and versatile platform by applying metabolic and enzyme engineering approaches. The present study aimed at drawing out the latent potential of this organism using genome-wide mutagenesis. To meet this goal, a transposon-based mutagenesis was carried out on E. coli, which was transformed to produce poly(lactate-co-3-hydroxybutyrate) from glucose. A high-throughput screening of polymer-accumulating cells on Nile red-containing plates isolated one mutant that produced 1.8-fold higher quantity of polymer without severe disadvantages in the cell growth and monomer composition of the polymer. The transposon was inserted into the locus within the gene encoding MtgA that takes part, as a non-lethal component, in the formation of the peptidoglycan backbone. Accordingly, the mtgA-deleted strain E. coli JW3175, which was a derivate of superior PHA-producing strain BW25113, was examined for polymer production, and exhibited an enhanced accumulation of the polymer (7.0 g/l) compared to the control (5.2 g/l). Interestingly, an enlargement in cell width associated with polymer accumulation was observed in this strain, resulting in a 1.6-fold greater polymer accumulation per cell compared to the control. This result suggests that the increase in volumetric capacity for accumulating intracellular material contributed to the enhanced polymer production. The mtgA deletion should be combined with conventional engineering approaches, and thus, is a promising strategy for improved production of intracellularly accumulated biopolymers. PMID:26039058

  20. Deletion of TGF-β signaling in myeloid cells enhances their anti-tumorigenic properties

    PubMed Central

    Novitskiy, Sergey V.; Pickup, Michael W.; Chytil, Anna; Polosukhina, Dina; Owens, Philip; Moses, Harold L.

    2012-01-01

    By crossing LysM-Cre and TGF-β type II receptor (Tgfbr2) floxed mice we achieved specific deletion of Tgfbr2 in myeloid cells (Tgfbr2MyeKO mice). S.c.-injected (LLC, EL4-OVA) and implanted (MMTV-PyMT) carcinoma cells grow slower in Tgfbr2MyeKO mice. The number of CD45+ cells in the tumor tissue was the same in both genotypes of mice, but upon analysis, the percentage of T cells (CD45+CD3+) in the KO mice was increased. By flow cytometry analysis, we did not detect any differences in the number and phenotype of TAMs, CD11b+Gr1+, and DCs in Tgfbr2MyeKO compared with Tgfbr2MyeWT mice. ELISA and qRT-PCR data showed differences in myeloid cell functions. In Tgfbr2MyeKO TAMs, TNF-α secretion was increased, basal IL-6 secretion was down-regulated, TGF-β did not induce any VEGF response, and there was decreased MMP9 and increased MMP2 and iNOS expression. TGF-β did not have any effect on CD11b+Gr1+ cells isolated from Tgfbr2MyeKO mice in the regulation of Arg, iNOS, VEGF, and CXCR4, and moreover, these cells have decreased suppressive activity relative to T cell proliferation. Also, we found that DCs from tumor tissue of Tgfbr2MyeKO mice have increased antigen-presented properties and an enhanced ability to stimulate antigen-specific T cell proliferation. We conclude that Tgfbr2 in myeloid cells has a negative role in the regulation of anti-tumorigenic functions of these cells, and deletion of this receptor decreases the suppressive function of CD11b+Gr1+ cells and increases antigen-presenting properties of DCs and anti-tumorigenic properties of TAMs. PMID:22685318

  1. The effects of mismatch repair and RAD1 genes on interchromosomal crossover recombination in Saccharomyces cerevisiae.

    PubMed

    Nicholson, Ainsley; Fabbri, Rebecca M; Reeves, Jason W; Crouse, Gray F

    2006-06-01

    We have previously shown that recombination between 400-bp substrates containing only 4-bp differences, when present in an inverted repeat orientation, is suppressed by >20-fold in wild-type strains of S. cerevisiae. Among the genes involved in this suppression were three genes involved in mismatch repair--MSH2, MSH3, and MSH6--and one in nucleotide excision repair, RAD1. We now report the involvement of these genes in interchromosomal recombination occurring via crossovers using these same short substrates. In these experiments, recombination was stimulated by a double-strand break generated by the HO endonuclease and can occur between completely identical (homologous) substrates or between nonidentical (homeologous) substrates. In addition, a unique feature of this system is that recombining DNA strands can be given a choice of either type of substrate. We find that interchromosomal crossover recombination with these short substrates is severely inhibited in the absence of MSH2, MSH3, or RAD1 and is relatively insensitive to the presence of mismatches. We propose that crossover recombination with these short substrates requires the products of MSH2, MSH3, and RAD1 and that these proteins have functions in recombination in addition to the removal of terminal nonhomology. We further propose that the observed insensitivity to homeology is a result of the difference in recombinational mechanism and/or the timing of the observed recombination events. These results are in contrast with those obtained using longer substrates and may be particularly relevant to recombination events between the abundant short repeated sequences that characterize the genomes of higher eukaryotes.

  2. Novel deletion mutants that enhance a distant upstream 5' splice in the E3 transcription unit of adenovirus 2.

    PubMed Central

    Deutscher, S L; Bhat, B M; Pursley, M H; Cladaras, C; Wold, W S

    1985-01-01

    Region E3 of adenovirus is a "complex" transcription unit: i.e. different mRNAs and proteins arise by differential RNA 3' end selection and differential splicing of the primary transcript. We are using viable virus mutants to understand the controls that dictate the specificity and efficiency of the RNA processing signals. We describe a novel class of deletion mutations that enhance a natural 5' splice site located approximately 740 nucleotides (nt) upstream. In particular, deletions within nt 1691-2044 in the E3 transcription unit result in a 5-fold enhancement of the 5' splice site at nt 951 (as reflected in steady-state mRNA). The effect is specific, because the deletions do not affect the 5' splice site at nt 372, and because deletions within nt 2044-2214 do not affect either the 951 or the 372 5' splice sites. As a consequence of the enhanced splicing at the 951 5' site, synthesis of the major E3 mRNA and the major E3 protein (gp19K) are dramatically reduced. At least one of the natural 3' splice sites, located at nt 2157, is the recipient of the enhanced splicing at the 951 5' splice site. We conclude that sequences located within nt 1691-2044 affect (probably in cis) splicing at the 951 5' splice site. We speculate that nt 1691-2044 includes a splicing control region which functions to suppress splicing at the 951 5' splice site. Images PMID:2412208

  3. Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

    PubMed

    Lyndaker, Amy M; Lim, Pei Xin; Mleczko, Joanna M; Diggins, Catherine E; Holloway, J Kim; Holmes, Rebecca J; Kan, Rui; Schlafer, Donald H; Freire, Raimundo; Cohen, Paula E; Weiss, Robert S

    2013-01-01

    The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

  4. A heterozygous deletion in the glutamate decarboxylase 67 gene enhances maternal and fetal stress vulnerability.

    PubMed

    Uchida, Taku; Oki, Yutaka; Yanagawa, Yuchio; Fukuda, Atsuo

    2011-04-01

    Both down-regulation of glutamate decarboxylase 67 (GAD67) and maternal exposure to severe stress during pregnancy can increase the risk of schizophrenia and related psychotic disorders in the offspring. To investigate a gene-environment interaction, we performed the restraint-and-light stress to pregnant GAD67-GFP knock-in (GAD67(+/GFP)) and wild-type (GAD67(+/+)) mice three times a day for 45 min per session during gestational day (G) 15.0-17.5. The stress hormone (corticosterone) level of pregnant GAD67(+/GFP) mice (the overall GABA content is reduced because of the destruction of one allele of the endogenous GAD67 gene) was higher than that of GAD67(+/+), even without stress. The fetal body weights (GAD67(+/+)) in the GAD67(+/GFP) mothers were lower than those in the GAD67(+/+) mothers. GAD67(+/GFP) fetuses exhibited higher corticosterone (CORT) levels than GAD67(+/+) fetuses, even in non-stressed GAD67(+/+) mothers. Fetal body weight-decreases and CORT-increases by maternal stress (GAD67(+/+) mother) were significantly more in the GAD67(+/GFP) fetuses than the GAD67(+/+) fetuses. These results indicate that a GAD67 heterozygous deletion itself enhances vulnerability by many aspects, e.g., maternal stress, maternity, and being in utero. Thus, an abnormality in GAD67 could interact with environmental risk factors of psychiatric disorders, including schizophrenia.

  5. Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature

    PubMed Central

    Benito-Sanz, Sara; Aza-Carmona, Miriam; Rodríguez-Estevez, Amaya; Rica-Etxebarria, Ixaso; Gracia, Ricardo; Campos-Barros, Ángel; Heath, Karen E

    2012-01-01

    Short stature homeobox-containing gene, MIM 312865 (SHOX) is located within the pseudoautosomal region 1 (PAR1) of the sex chromosomes. Mutations in SHOX or its downstream transcriptional regulatory elements represent the underlying molecular defect in ∼60% of Léri-Weill dyschondrosteosis (LWD) and ∼5–15% of idiopathic short stature (ISS) patients. Recently, three novel enhancer elements have been identified upstream of SHOX but to date, no PAR1 deletions upstream of SHOX have been observed that only encompass these enhancers in LWD or ISS patients. We set out to search for genetic alterations of the upstream SHOX regulatory elements in 63 LWD and 100 ISS patients with no known alteration in SHOX or the downstream enhancer regions using a specifically designed MLPA assay, which covers the PAR1 upstream of SHOX. An upstream SHOX deletion was identified in an ISS proband and her affected father. The deletion was confirmed and delimited by array-CGH, to extend ∼286 kb. The deletion included two of the upstream SHOX enhancers without affecting SHOX. The 13.3-year-old proband had proportionate short stature with normal GH and IGF-I levels. In conclusion, we have identified the first PAR1 deletion encompassing only the upstream SHOX transcription regulatory elements in a family with ISS. The loss of these elements may result in SHOX haploinsufficiency because of decreased SHOX transcription. Therefore, this upstream region should be included in the routine analysis of PAR1 in patients with LWD, LMD and ISS. PMID:22071895

  6. Deleted in Azoospermia-Like Enhances In Vitro Derived Porcine Germ Cell Formation and Meiosis

    PubMed Central

    Park, Bong-Wook; Shen, Wei; Linher-Melville, Katja

    2013-01-01

    Evidence supporting that deleted in azoospermia-like (DAZL) plays a key role during gametogenesis and meiosis continues to emerge. Our study aimed to determine whether overexpression of DAZL using a lentiviral approach in a somatic stem cell to germ cell in vitro differentiation culture could enhance the formation of primordial germ cell-like cells (PLCs) and oocyte-like cells (OLCs). Introduction of DAZL at the beginning of induced differentiation significantly increased the formation of Fragilis-positive PLCs, which was independent of mitotic proliferation. In addition, mRNA levels of the germ cell markers Oct4, Stella, and Vasa were also higher in the DAZL-transduced group and suppressed when DAZL was knocked down using small interference RNA. At later stages of differentiation, the expression of several genes associated with meiosis, including Scp3, Dmc1, Rec8, and Stra8, was determined to be significantly higher when DAZL was overexpressed, which was abrogated by its knockdown. Exogenous introduction of DAZL also increased the protein levels of SCP3 and VASA, which again was reversed by its knockdown. Although not a common phenomenon in the in vitro differentiation system, the percentage of SCP3-positive cells displaying meiotic chromosome patterns in the DAZL-transduced group was higher than in the control, as was the overall percentage of OLCs that were generated. The introduction of factors such as DAZL into a stem cell-to-germ cell differentiation culture may provide an opportunity to better understand the key genes and their interactions during gametogenesis, also providing a means to enhance the generation of germ cells in vitro. PMID:23259838

  7. Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production.

    PubMed

    Trikka, Fotini A; Nikolaidis, Alexandros; Athanasakoglou, Anastasia; Andreadelli, Aggeliki; Ignea, Codruta; Kotta, Konstantia; Argiriou, Anagnostis; Kampranis, Sotirios C; Makris, Antonios M

    2015-04-24

    Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system. Aiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation. Using an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.

  8. Deletion of Virus-specific T-cells Enhances Remyelination in a Model of Multiple Sclerosis.

    PubMed

    Denic, Aleksandar; Wootla, Bharath; Zoecklein, Laurie; Rodriguez, Moses

    2014-01-01

    We used transgenic expression of capsid antigens to Theiler's murine encephalomyelitis virus (TMEV) to study how the immune response to VP1 and VP2 influences spinal cord demyelination, remyelination and axonal loss during the acute and chronic phases of infection. Expression from birth of capsid antigen under the ubiquitin promoter resulted in tolerance to the antigen and absence of an immune response to the respective capsid antigen following virus infection. The transgenic mice were crossed to B10.Q mice normally susceptible to demyelination but which, when compared to FVB mice of the same H2 (q) haplotype, show poor remyelination. The major finding in this study was that VP1+ and VP2+ animals featured more remyelination at all three chronic time points (90, 180 and 270 dpi) than transgene-negative controls. Interestingly, at 270 dpi, remyelination in VP1+ mice tended to be higher and more complete than that in VP2+ mice. Compared with transgene- negative controls, VP1+ and VP2+ animals showed similar demyelination in but less only late in the disease (270 dpi). The number of mid-thoracic axons at the last time point correlated with the levels of remyelination. The increase in number of axons in VP1+ mice with remyelination was driven by counts in medium- and large-caliber axons. This study supports the hypothesis that expression of viral capsid proteins as self and subsequent genetic deletion of capsid-specific T cells influences the extent of spinal cord remyelination following Theiler's virus-induced demyelination. We propose that VP1- and, to a lesser extent, VP2-specific CD8(+) T cells limit and/or prevent the naturally occurring process of remyelination. This finding may have relevance to human multiple sclerosis, as targeted removal of CD8(+) T cells specific for a yet-to-be-discovered causative peptide may enhance remyelination and prevent axonal loss in patients.

  9. Enhanced Maternal Origin of the 22q11.2 Deletion in Velocardiofacial and DiGeorge Syndromes

    PubMed Central

    Delio, Maria; Guo, Tingwei; McDonald-McGinn, Donna M.; Zackai, Elaine; Herman, Sean; Kaminetzky, Mark; Higgins, Anne Marie; Coleman, Karlene; Chow, Carolyn; Jarlbrzkowski, Maria; Bearden, Carrie E.; Bailey, Alice; Vangkilde, Anders; Olsen, Line; Olesen, Charlotte; Skovby, Flemming; Werge, Thomas M.; Templin, Ludivine; Busa, Tiffany; Philip, Nicole; Swillen, Ann; Vermeesch, Joris R.; Devriendt, Koen; Schneider, Maude; Dahoun, Sophie; Eliez, Stephan; Schoch, Kelly; Hooper, Stephen R.; Shashi, Vandana; Samanich, Joy; Marion, Robert; van Amelsvoort, Therese; Boot, Erik; Klaassen, Petra; Duijff, Sasja N.; Vorstman, Jacob; Yuen, Tracy; Silversides, Candice; Chow, Eva; Bassett, Anne; Frisch, Amos; Weizman, Abraham; Gothelf, Doron; Niarchou, Maria; van den Bree, Marianne; Owen, Michael J.; Suñer, Damian Heine; Andreo, Jordi Rosell; Armando, Marco; Vicari, Stefano; Digilio, Maria Cristina; Auton, Adam; Kates, Wendy R.; Wang, Tao; Shprintzen, Robert J.; Emanuel, Beverly S.; Morrow, Bernice E.

    2013-01-01

    Velocardiofacial and DiGeorge syndromes, also known as 22q11.2 deletion syndrome (22q11DS), are congenital-anomaly disorders caused by a de novo hemizygous 22q11.2 deletion mediated by meiotic nonallelic homologous recombination events between low-copy repeats, also known as segmental duplications. Although previous studies exist, each was of small size, and it remains to be determined whether there are parent-of-origin biases for the de novo 22q11.2 deletion. To address this question, we genotyped a total of 389 DNA samples from 22q11DS-affected families. A total of 219 (56%) individuals with 22q11DS had maternal origin and 170 (44%) had paternal origin of the de novo deletion, which represents a statistically significant bias for maternal origin (p = 0.0151). Combined with many smaller, previous studies, 465 (57%) individuals had maternal origin and 345 (43%) had paternal origin, amounting to a ratio of 1.35 or a 35% increase in maternal compared to paternal origin (p = 0.000028). Among 1,892 probands with the de novo 22q11.2 deletion, the average maternal age at time of conception was 29.5, and this is similar to data for the general population in individual countries. Of interest, the female recombination rate in the 22q11.2 region was about 1.6–1.7 times greater than that for males, suggesting that for this region in the genome, enhanced meiotic recombination rates, as well as other as-of-yet undefined 22q11.2-specific features, could be responsible for the observed excess in maternal origin. PMID:23453669

  10. Deletion of neuropilin 2 enhances detrusor contractility following bladder outlet obstruction

    PubMed Central

    Vasquez, Evalynn; Cristofaro, Vivian; Lukianov, Stefan; Burkhard, Fiona C.; Monastyrskaya, Katia; Bielenberg, Diane R.; Sullivan, Maryrose P.; Adam, Rosalyn M.

    2017-01-01

    Chronic urethral obstruction and the ensuing bladder wall remodeling can lead to diminished bladder smooth muscle (BSM) contractility and debilitating lower urinary tract symptoms. No effective pharmacotherapy exists to restore BSM contractile function. Neuropilin 2 (Nrp2) is a transmembrane protein that is highly expressed in BSM. Nrp2 deletion in mice leads to increased BSM contraction. We determined whether genetic ablation of Nrp2 could restore BSM contractility following obstruction. Partial bladder outlet obstruction (pBOO) was created by urethral occlusion in mice with either constitutive and ubiquitous, or inducible smooth muscle–specific deletion of Nrp2, and Nrp2-intact littermates. Mice without obstruction served as additional controls. Contractility was measured by isometric tension testing. Nrp2 deletion prior to pBOO increased force generation in BSM 4 weeks following surgery. Deletion of Nrp2 in mice already subjected to pBOO for 4 weeks showed increased contractility of tissues tested 6 weeks after surgery compared with nondeleted controls. Assessment of tissues from patients with urodynamically defined bladder outlet obstruction revealed reduced NRP2 levels in obstructed bladders with compensated compared with decompensated function, relative to asymptomatic controls. We conclude that downregulation of Nrp2 promotes BSM force generation. Neuropilin 2 may represent a novel target to restore contractility following obstruction. PMID:28194441

  11. Deletion of neuropilin 2 enhances detrusor contractility following bladder outlet obstruction.

    PubMed

    Vasquez, Evalynn; Cristofaro, Vivian; Lukianov, Stefan; Burkhard, Fiona C; Gheinani, Ali Hashemi; Monastyrskaya, Katia; Bielenberg, Diane R; Sullivan, Maryrose P; Adam, Rosalyn M

    2017-02-09

    Chronic urethral obstruction and the ensuing bladder wall remodeling can lead to diminished bladder smooth muscle (BSM) contractility and debilitating lower urinary tract symptoms. No effective pharmacotherapy exists to restore BSM contractile function. Neuropilin 2 (Nrp2) is a transmembrane protein that is highly expressed in BSM. Nrp2 deletion in mice leads to increased BSM contraction. We determined whether genetic ablation of Nrp2 could restore BSM contractility following obstruction. Partial bladder outlet obstruction (pBOO) was created by urethral occlusion in mice with either constitutive and ubiquitous, or inducible smooth muscle-specific deletion of Nrp2, and Nrp2-intact littermates. Mice without obstruction served as additional controls. Contractility was measured by isometric tension testing. Nrp2 deletion prior to pBOO increased force generation in BSM 4 weeks following surgery. Deletion of Nrp2 in mice already subjected to pBOO for 4 weeks showed increased contractility of tissues tested 6 weeks after surgery compared with nondeleted controls. Assessment of tissues from patients with urodynamically defined bladder outlet obstruction revealed reduced NRP2 levels in obstructed bladders with compensated compared with decompensated function, relative to asymptomatic controls. We conclude that downregulation of Nrp2 promotes BSM force generation. Neuropilin 2 may represent a novel target to restore contractility following obstruction.

  12. Enhancing the peroxidatic activity of KatG by deletion mutagenesis.

    PubMed

    Kudalkar, Shalley N; Campbell, Robert A; Li, Yongjiang; Varnado, Cornelius L; Prescott, Corey; Goodwin, Douglas C

    2012-11-01

    Catalase-peroxidase (KatG) enzymes use a peroxidase active site to facilitate robust catalase activity, an ability all other members of its superfamily lack. KatG's have a Met-Tyr-Trp covalent adduct that is essential for catalatic but not peroxidatic turnover. The tyrosine (Y226 in E. coli KatG) is supplied by a large loop (LL1) that is absent from all other plant peroxidases. Elimination of Y226 from the KatG structure, either by site directed mutagenesis (i.e., Y226F KatG) or by deletion of larger portions of LL1 invariably eliminates catalase activity, but deletion variants were substantially more active as peroxidases, up to an order of magnitude. Moreover, the deletion variants were more resistant to H(2)O(2)-dependent inactivation than Y226F KatG. Stopped-flow evaluation of reactions of H(2)O(2) with Y226F KatG and the most peroxidase active deletion variant (KatG[Δ209-228]) produced highly similar rate constants for formation of compounds I and II, and about a four-fold faster formation of compound III for the deletion variant as opposed to Y226F. Conversely, single turnover experiments showed a 60-fold slower return of Y226F KatG to its ferric state in the presence of the exogenous electron donor 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) than was determined for KatG(Δ209-228). Our data suggest that the peroxidatic output of KatG cannot be optimized simply by elimination of catalase activity alone, but also requires modifications that increase electron transfer between exogenous electron donors and the heme prosthetic group. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Enhanced leavening ability of baker's yeast by overexpression of SNR84 with PGM2 deletion.

    PubMed

    Lin, Xue; Zhang, Cui-Ying; Bai, Xiao-Wen; Xiao, Dong-Guang

    2015-06-01

    Dough-leavening ability is one of the main aspects considered when selecting a baker's yeast strain for baking industry. Generally, modification of maltose metabolic pathway and known regulatory networks of maltose metabolism were used to increase maltose metabolism to improve leavening ability in lean dough. In this study, we focus on the effects of PGM2 (encoding for the phosphoglucomutase) and SNR84 (encoding for the H/ACA snoRNA) that are not directly related to both the maltose metabolic pathway and known regulatory networks of maltose metabolism on the leavening ability of baker's yeast in lean dough. The results show that the modifications on PGM2 and/or SNR84 are effective ways in improving leavening ability of baker's yeast in lean dough. Deletion of PGM2 decreased cellular glucose-1-phosphate and overexpression of SNR84 increased the maltose permease activity. These changes resulted in 11, 19 and 21% increases of the leavening ability for PGM2 deletion, SNR84 overexpression and SNR84 overexpression combining deleted PGM2, respectively.

  14. Tph2 gene deletion enhances amphetamine-induced hypermotility: effect of 5-HT restoration and role of striatal noradrenaline release.

    PubMed

    Carli, Mirjana; Kostoula, Chrysaugi; Sacchetti, Giuseppina; Mainolfi, Pierangela; Anastasia, Alessia; Villani, Claudia; Invernizzi, Roberto William

    2015-11-01

    Variants of tryptophan hydroxylase-2 (Tph2), the gene encoding enzyme responsible for the synthesis of brain serotonin (5-HT), have been associated with neuropsychiatric disorders, substance abuse and addiction. This study assessed the effect of Tph2 gene deletion on motor behavior and found that motor activity induced by 2.5 and 5 mg/kg amphetamine was enhanced in Tph2(-/-) mice. Using the in vivo microdialysis technique we found that the ability of amphetamine to stimulate noradrenaline (NA) release in the striatum was reduced by about 50% in Tph2(-/-) mice while the release of dopamine (DA) was not affected. Tph2 deletion did not affect the release of NA and DA in the prefrontal cortex. The role of endogenous 5-HT in enhancing the effect of amphetamine was confirmed showing that treatment with the 5-HT precursor 5-hydroxytryptophan (10 mg/kg) restored tissue and extracellular levels of brain 5-HT and the effects of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. Treatment with the NA precursor dihydroxyphenylserine (400 mg/kg) was sufficient to restore the effect of amphetamine on striatal NA release and motor activity in Tph2(-/-) mice. These findings indicate that amphetamine-induced hyperactivity is attenuated by endogenous 5-HT through the inhibition of striatal NA release. Tph2(-/-) mice may be a useful preclinical model to assess the role of 5-HT-dependent mechanisms in the action of psychostimulants. Acute sensitivity to the motor effects of amphetamine has been associated to increased risk of psychostimulant abuse. Here, we show that deletion of Tph2, the gene responsible for brain 5-HT synthesis, enhances the motor effect of amphetamine in mice through the inhibition of striatal NA release. This suggests that Tph2(-/-) mice is a useful preclinical model to assess the role of 5-HT-dependent mechanisms in psychostimulants action. Tph2, tryptophan hydroxylase-2.

  15. "Black holes" and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli.

    PubMed

    Maurelli, A T; Fernández, R E; Bloch, C A; Rode, C K; Fasano, A

    1998-03-31

    Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in approximately 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these "black holes," deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

  16. ``Black Holes" and Bacterial Pathogenicity: A Large Genomic Deletion that Enhances the Virulence of Shigella spp. and Enteroinvasive Escherichia coli

    NASA Astrophysics Data System (ADS)

    Maurelli, Anthony T.; Fernandez, Reinaldo E.; Bloch, Craig A.; Rode, Christopher K.; Fasano, Alessio

    1998-03-01

    Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylate (LDC) activity is present in ≈ 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these ``black holes,'' deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

  17. Deletion of a Vibrio cholerae ClC Channel Results in Acid Sensitivity and Enhanced Intestinal Colonization

    PubMed Central

    Ding, Yanpeng; Waldor, Matthew K.

    2003-01-01

    ClC chloride channels are found in all three kingdoms of life though little is known about their functions in prokaryotes. Here we investigated the role of a Vibrio cholerae ClC channel in acid resistance and intestinal colonization. The putative V. cholerae ClC channel was found to confer mild resistance to acid when pH was adjusted with HCl, but not with other acids. Surprisingly, a ClC channel deletion mutant exhibited enhanced intestinal colonization in infant mice. PMID:12819118

  18. Enhancement or Attenuation of Disease by Deletion of Genes from Citrus Tristeza Virus

    PubMed Central

    Tatineni, Satyanarayana

    2012-01-01

    Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9Δp33 and CTV9Δp33Δp18Δp13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development. PMID:22593155

  19. Enhancement or attenuation of disease by deletion of genes from Citrus tristeza virus.

    PubMed

    Tatineni, Satyanarayana; Dawson, William O

    2012-08-01

    Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9Δp33 and CTV9Δp33Δp18Δp13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development.

  20. Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells.

    PubMed

    Ojima, Yoshihiro; Mohanadas, Thivagaran; Kitamura, Kosei; Nunogami, Shota; Yajima, Reiki; Taya, Masahito

    2017-04-01

    Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

  1. Ager Deletion Enhances Ischemic Muscle Inflammation, Angiogenesis, and Blood Flow Recovery in Diabetic Mice.

    PubMed

    López-Díez, Raquel; Shen, Xiaoping; Daffu, Gurdip; Khursheed, Md; Hu, Jiyuan; Song, Fei; Rosario, Rosa; Xu, Yunlu; Li, Qing; Xi, Xiangmei; Zou, Yu Shan; Li, Huilin; Schmidt, Ann Marie; Yan, Shi Fang

    2017-08-01

    Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block angiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated angiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1 (early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, angiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6C(hi) monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. These findings indicate that RAGE attenuates adaptive inflammation in hindlimb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic peripheral vascular disease. © 2017 American Heart Association, Inc.

  2. Adipocyte-specific deletion of Ip6k1 reduces diet-induced obesity by enhancing AMPK-mediated thermogenesis.

    PubMed

    Zhu, Qingzhang; Ghoshal, Sarbani; Rodrigues, Ana; Gao, Su; Asterian, Alice; Kamenecka, Theodore M; Barrow, James C; Chakraborty, Anutosh

    2016-11-01

    Enhancing energy expenditure (EE) is an attractive strategy to combat obesity and diabetes. Global deletion of Ip6k1 protects mice from diet-induced obesity (DIO) and insulin resistance, but the tissue-specific mechanism by which IP6K1 regulates body weight is unknown. Here, we have demonstrated that IP6K1 regulates fat accumulation by modulating AMPK-mediated adipocyte energy metabolism. Cold exposure led to downregulation of Ip6k1 in murine inguinal and retroperitoneal white adipose tissue (IWAT and RWAT) depots. Adipocyte-specific deletion of Ip6k1 (AdKO) enhanced thermogenic EE, which protected mice from high-fat diet-induced weight gain at ambient temperature (23°C), but not at thermoneutral temperature (30°C). AdKO-induced increases in thermogenesis also protected mice from cold-induced decreases in body temperature. UCP1, PGC1α, and other markers of browning and thermogenesis were elevated in IWAT and RWAT of AdKO mice. Cold-induced activation of sympathetic signaling was unaltered, whereas AMPK was enhanced, in AdKO IWAT. Moreover, beige adipocytes from AdKO IWAT displayed enhanced browning, which was diminished by AMPK depletion. Furthermore, we determined that IP6 and IP6K1 differentially regulate upstream kinase-mediated AMPK stimulatory phosphorylation in vitro. Finally, treating mildly obese mice with the IP6K inhibitor TNP enhanced thermogenesis and inhibited progression of DIO. Thus, IP6K1 regulates energy metabolism via a mechanism that could potentially be targeted in obesity.

  3. Adipocyte-specific deletion of Ip6k1 reduces diet-induced obesity by enhancing AMPK-mediated thermogenesis

    PubMed Central

    Zhu, Qingzhang; Ghoshal, Sarbani; Rodrigues, Ana; Gao, Su; Asterian, Alice; Kamenecka, Theodore M.; Barrow, James C.

    2016-01-01

    Enhancing energy expenditure (EE) is an attractive strategy to combat obesity and diabetes. Global deletion of Ip6k1 protects mice from diet-induced obesity (DIO) and insulin resistance, but the tissue-specific mechanism by which IP6K1 regulates body weight is unknown. Here, we have demonstrated that IP6K1 regulates fat accumulation by modulating AMPK-mediated adipocyte energy metabolism. Cold exposure led to downregulation of Ip6k1 in murine inguinal and retroperitoneal white adipose tissue (IWAT and RWAT) depots. Adipocyte-specific deletion of Ip6k1 (AdKO) enhanced thermogenic EE, which protected mice from high-fat diet–induced weight gain at ambient temperature (23°C), but not at thermoneutral temperature (30°C). AdKO-induced increases in thermogenesis also protected mice from cold-induced decreases in body temperature. UCP1, PGC1α, and other markers of browning and thermogenesis were elevated in IWAT and RWAT of AdKO mice. Cold-induced activation of sympathetic signaling was unaltered, whereas AMPK was enhanced, in AdKO IWAT. Moreover, beige adipocytes from AdKO IWAT displayed enhanced browning, which was diminished by AMPK depletion. Furthermore, we determined that IP6 and IP6K1 differentially regulate upstream kinase-mediated AMPK stimulatory phosphorylation in vitro. Finally, treating mildly obese mice with the IP6K inhibitor TNP enhanced thermogenesis and inhibited progression of DIO. Thus, IP6K1 regulates energy metabolism via a mechanism that could potentially be targeted in obesity. PMID:27701146

  4. FOXO1 deletion reduces dendritic cell function and enhances susceptibility to periodontitis.

    PubMed

    Xiao, Wenmei; Dong, Guangyu; Pacios, Sandra; Alnammary, Maher; Barger, Laura A; Wang, Yu; Wu, Yingying; Graves, Dana T

    2015-04-01

    The host response plays both protective and destructive roles in periodontitis. FOXO1 is a transcription factor that is activated in dendritic cells (DCs), but its function in vivo has not been examined. We investigated the role of FOXO1 in activating DCs in experimental (CD11c.Cre(+).FOXO1(L/L)) compared with matched control mice (CD11c.Cre(-).FOXO1(L/L)) in response to oral pathogens. Lineage-specific FOXO1 deletion reduced the recruitment of DCs to oral mucosal epithelium by approximately 40%. FOXO1 was needed for expression of genes that regulate migration, including integrins αν and β3 and matrix metalloproteinase-2. Ablation of FOXO1 in DCs significantly decreased IL-12 produced by DCs in mucosal surfaces. Moreover, FOXO1 deletion reduced migration of DCs to lymph nodes, reduced capacity of DCs to induce formation of plasma cells, and reduced production of bacteria-specific antibody. The decrease in DC function in the experimental mice led to increased susceptibility to periodontitis through a mechanism that involved a compensatory increase in osteoclastogenic factors, IL-1β, IL-17, and RANKL. Thus, we reveal a critical role for FOXO1 in DC recruitment to oral mucosal epithelium and activation of adaptive immunity induced by oral inoculation of bacteria. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  5. Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production.

    PubMed

    Jung, Moo-Young; Ng, Chiam Yu; Song, Hyohak; Lee, Jinwon; Oh, Min-Kyu

    2012-07-01

    2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

  6. FOXO1 Deletion Reduces Dendritic Cell Function and Enhances Susceptibility to Periodontitis

    PubMed Central

    Xiao, Wenmei; Dong, Guangyu; Pacios, Sandra; Alnammary, Maher; Barger, Laura A.; Wang, Yu; Wu, Yingying; Graves, Dana T.

    2016-01-01

    The host response plays both protective and destructive roles in periodontitis. FOXO1 is a transcription factor that is activated in dendritic cells (DCs), but its function in vivo has not been examined. We investigated the role of FOXO1 in activating DCs in experimental (CD11c.Cre+.FOXO1L/L) compared with matched control mice (CD11c.Cre−.FOXO1L/L) in response to oral pathogens. Lineage-specific FOXO1 deletion reduced the recruitment of DCs to oral mucosal epithelium by approximately 40%. FOXO1 was needed for expression of genes that regulate migration, including integrins αν and β3 and matrix metalloproteinase-2. Ablation of FOXO1 in DCs significantly decreased IL-12 produced by DCs in mucosal surfaces. Moreover, FOXO1 deletion reduced migration of DCs to lymph nodes, reduced capacity of DCs to induce formation of plasma cells, and reduced production of bacteria-specific antibody. The decrease in DC function in the experimental mice led to increased susceptibility to periodontitis through a mechanism that involved a compensatory increase in osteoclastogenic factors, IL-1β, IL-17, and RANKL. Thus, we reveal a critical role for FOXO1 in DC recruitment to oral mucosal epithelium and activation of adaptive immunity induced by oral inoculation of bacteria. PMID:25794707

  7. Induction of Ty Recombination in Yeast by Cdna and Transcription: Role of the Rad1 and Rad52 Genes

    PubMed Central

    Nevo-Caspi, Y.; Kupiec, M.

    1996-01-01

    In the yeast Saccharomyces cerevisiae ectopic recombination has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family. Little is known about the mechanism(s) that prevent recombination between repeated sequences. We have previously shown that nonreciprocal recombination (gene conversion) of a genetically marked Ty can be induced either by the presence of high levels of Ty cDNA or by transcription of the marked Ty from a GAL1 promoter. These two kinds of induction act in a synergistic manner. To further characterize these two kinds of Ty recombination, we have investigated the role played by the RAD52 and RAD1 genes. We have found that the RAD52 and RAD1 gene products are essential to carry out transcription-induced Ty conversion whereas cDNA-mediated conversion can take place in their absence. PMID:8913740

  8. Structural and functional analyses of an archaeal XPF/Rad1/Mus81 nuclease: asymmetric DNA binding and cleavage mechanisms.

    PubMed

    Nishino, Tatsuya; Komori, Kayoko; Ishino, Yoshizumi; Morikawa, Kosuke

    2005-08-01

    XPF/Rad1/Mus81/Hef proteins recognize and cleave branched DNA structures. XPF and Rad1 proteins cleave the 5' side of nucleotide excision repair bubble, while Mus81 and Hef cleave similar sites of the nicked Holliday junction, fork, or flap structure. These proteins all function as dimers and consist of catalytic and helix-hairpin-helix DNA binding (HhH) domains. We have determined the crystal structure of the HhH domain of Pyrococcus furiosus Hef nuclease (HefHhH), which revealed the distinct mode of protein dimerization. Our structural and biochemical analyses also showed that each of the catalytic and HhH domains binds to distinct regions within the fork-structured DNA: each HhH domain from two separate subunits asymmetrically binds to the arm region, while the catalytic domain binds near the junction center. Upon binding to DNA, Hef nuclease disrupts base pairs near the cleavage site. It is most likely that this bipartite binding mode is conserved in the XPF/Rad1/Mus81 nuclease family.

  9. Arginase 2 deletion leads to enhanced M1 macrophage activation and upregulated polyamine metabolism in response to Helicobacter pylori infection.

    PubMed

    Hardbower, Dana M; Asim, Mohammad; Murray-Stewart, Tracy; Casero, Robert A; Verriere, Thomas; Lewis, Nuruddeen D; Chaturvedi, Rupesh; Piazuelo, M Blanca; Wilson, Keith T

    2016-10-01

    We reported that arginase 2 (ARG2) deletion results in increased gastritis and decreased bacterial burden during Helicobacter pylori infection in mice. Our studies implicated a potential role for inducible nitric oxide (NO) synthase (NOS2), as Arg2 (-/-) mice exhibited increased NOS2 levels in gastric macrophages, and NO can kill H. pylori. We now bred Arg2 (-/-) to Nos2 (-/-) mice, and infected them with H. pylori. Compared to wild-type mice, both Arg2 (-/-) and Arg2 (-/-) ;Nos2 (-/-) mice exhibited increased gastritis and decreased colonization, the latter indicating that the effect of ARG2 deletion on bacterial burden was not mediated by NO. While Arg2 (-/-) mice demonstrated enhanced M1 macrophage activation, Nos2 (-/-) and Arg2 (-/-) ;Nos2 (-/-) mice did not demonstrate these changes, but exhibited increased CXCL1 and CXCL2 responses. There was an increased expression of the Th1/Th17 cytokines, interferon gamma and interleukin 17, in gastric tissues and splenic T-cells from Arg2 (-/-), but not Nos2 (-/-) or Arg2 (-/-) ;Nos2 (-/-) mice. Gastric tissues from infected Arg2 (-/-) mice demonstrated increased expression of arginase 1, ornithine decarboxylase, adenosylmethionine decarboxylase 1, spermidine/spermine N (1)-acetyltransferase 1, and spermine oxidase, along with increased spermine levels. These data indicate that ARG2 deletion results in compensatory upregulation of gastric polyamine synthesis and catabolism during H. pylori infection, which may contribute to increased gastric inflammation and associated decreased bacterial load. Overall, the finding of this study is that ARG2 contributes to the immune evasion of H. pylori by restricting M1 macrophage activation and polyamine metabolism.

  10. Deletion of ldhA and aldH genes in Klebsiella pneumoniae to enhance 1,3-propanediol production.

    PubMed

    Chen, Lifei; Ma, Chunling; Wang, Ruiming; Yang, Jianlou; Zheng, Haijie

    2016-10-01

    To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia. Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains. Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

  11. Genome-wide screening of transcription factor deletion targets in Escherichia coli for enhanced production of lactate-based polyesters.

    PubMed

    Kadoya, Ryosuke; Kodama, Yu; Matsumoto, Ken'ichiro; Ooi, Toshihiko; Taguchi, Seiichi

    2017-05-01

    Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (ΔpdhR, ΔcspG, ΔyneJ, ΔchbR, ΔyiaU, ΔcreB, ΔygfI and ΔnanK) accumulated greater amount of polymer (6.2-10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1-1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34-0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions.

  12. Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice

    PubMed Central

    Sastre, Cristina; Fernández-Laso, Valvanera; Madrigal-Matute, Julio; Muñoz-García, Begoña; Moreno, Juan A; Pastor-Vargas, Carlos; Llamas-Granda, Patricia; Burkly, Linda C; Egido, Jesús; Martín-Ventura, Jose L; Blanco-Colio, Luis M

    2014-01-01

    Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12−/−ApoE−/−) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12−/−ApoE−/− or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12+/+ApoE−/− control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12−/−ApoE−/−. A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12+/+ApoE−/− mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12−/−ApoE−/− or anti-TWEAK treatment in TNFSF12+/+ApoE−/− mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish

  13. The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining1[OPEN

    PubMed Central

    Tang, Ding; Shen, Yi; Chen, Xiaojun; Ji, Jianhui; Du, Guijie; Li, Yafei; Cheng, Zhukuan

    2016-01-01

    During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1. In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice. PMID:27512017

  14. CCAAT/enhancer binding protein {beta} deletion increases mitochondrial function and protects mice from LXR-induced hepatic steatosis

    SciTech Connect

    Rahman, Shaikh M.; Choudhury, Mahua; Janssen, Rachel C.; Baquero, Karalee C.; Miyazaki, Makoto; Friedman, Jacob E.

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer LXR agonist activation increases liver TG accumulation by increasing lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta}{sup -/-} mouse prevents LXR activation-mediated induction of hepatic lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta} deletion increases mitochondrial transport chain function. Black-Right-Pointing-Pointer Beneficial effects of LXR activation on liver cholesterol metabolism did not change. Black-Right-Pointing-Pointer C/EBP{beta} inhibition might have important therapeutic potential. -- Abstract: Drugs designed specifically to activate liver X receptors (LXRs) have beneficial effects on lowering cholesterol metabolism and inflammation but unfortunately lead to severe hepatic steatosis. The transcription factor CCAAT/enhancer binding protein beta (C/EBP{beta}) is an important regulator of liver gene expression but little is known about its involvement in LXR-based steatosis and cholesterol metabolism. The present study investigated the role of C/EBP{beta} expression in LXR agonist (T0901317)-mediated alteration of hepatic triglyceride (TG) and lipogenesis in mice. C/EBP{beta} deletion in mice prevented LXR agonist-mediated induction of lipogenic gene expression in liver in conjunction with significant reduction of liver TG accumulation. Surprisingly, C/EBP{beta}{sup -/-} mice showed a major increase in liver mitochondrial electron chain function compared to WT mice. Furthermore, LXR activation in C/EBP{beta}{sup -/-} mice increased the expression of liver ATP-binding cassette transporter ABCG1, a gene implicated in cholesterol efflux and reducing blood levels of total and LDL-cholesterol. Together, these findings establish a central role for C/EBP{beta} in the LXR-mediated steatosis and mitochondrial function, without impairing the influence of LXR activation on lowering LDL and increasing HDL-cholesterol. Inactivation of C/EBP{beta} might therefore be an important therapeutic strategy to prevent LXR

  15. Conditional genetic deletion of PTEN after a spinal cord injury enhances regenerative growth of CST axons and motor function recovery in mice.

    PubMed

    Danilov, Camelia A; Steward, Oswald

    2015-04-01

    Previous studies indicate that conditional genetic deletion of phosphatase and tensin homolog (PTEN) in neonatal mice enhances the ability of axons to regenerate following spinal cord injury (SCI) in adults. Here, we assessed whether deleting PTEN in adult neurons post-SCI is also effective, and whether enhanced regenerative growth is accompanied by enhanced recovery of voluntary motor function. PTEN(loxP/loxP) mice received moderate contusion injuries at cervical level 5 (C5). One group received unilateral injections of adeno-associated virus expressing CRE (AAV-CRE) into the sensorimotor cortex; controls received a vector expressing green fluorescent protein (AAV-GFP) or injuries only (no vector injections). Forelimb function was tested for 14weeks post-SCI using a grip strength meter (GSM) and a hanging task. The corticospinal tract (CST) was traced by injecting mini-ruby BDA into the sensorimotor cortex. Forelimb gripping ability was severely impaired immediately post-SCI but recovered slowly over time. The extent of recovery was significantly greater in PTEN-deleted mice in comparison to either the AAV-GFP group or the injury only group. BDA tract tracing revealed significantly higher numbers of BDA-labeled axons in caudal segments in the PTEN-deleted group compared to control groups. In addition, in the PTEN-deleted group, there were exuberant collaterals extending from the main tract rostral to the lesion and into and around the scar tissue at the injury site. These results indicate that PTEN deletion in adult mice shortly post-SCI can enhance regenerative growth of CST axons and forelimb motor function recovery.

  16. Conditional genetic deletion of PTEN after a spinal cord injury enhances regenerative growth of CST axons and motor function recovery in mice

    PubMed Central

    Danilov, Camelia A.; Steward, Oswald

    2015-01-01

    Previous studies indicate that conditional genetic deletion of phosphatase and tensin homolog (PTEN) in neonatal mice enhances the ability of axons to regenerate following spinal cord injury (SCI) in adults. Here, we assessed whether deleting PTEN in adult neurons post-SCI is also effective, and whether enhanced regenerative growth is accompanied by enhanced recovery of voluntary motor function. PTENloxP/loxP mice received moderate contusion injuries at cervical level 5 (C5). One group received unilateral injections of adeno-associated virus expressing CRE (AAV-CRE) into the sensorimotor cortex; controls received a vector expressing green fluorescent protein (AAV-GFP) or injuries only (no vector injections). Forelimb function was tested for 14 weeks post-SCI using a grip strength meter (GSM) and a hanging task. The corticospinal tract (CST) was traced by injecting mini-ruby BDA into the sensorimotor cortex. Forelimb gripping ability was severely impaired immediately post-SCI but recovered slowly over time. The extent of recovery was significantly greater in PTEN-deleted mice in comparison to either the AAV-GFP group or the injury only group. BDA tract tracing revealed significantly higher numbers of BDA-labeled axons in caudal segments in the PTEN-deleted group compared to control groups. In addition, in the PTEN-deleted group, there were exuberant collaterals extending from the main tract rostral to the lesion, into and around the scar tissue at the injury site. These results indicate that PTEN deletion in adult mice shortly post-SCI can enhance regenerative growth of CST axons and forelimb motor function recovery. PMID:25704959

  17. Adipose tissue hyperplasia with enhanced adipocyte-derived stem cell activity in Tc1(C8orf4)-deleted mice

    PubMed Central

    Jang, Hayoung; Kim, Minsung; Lee, Soyoung; Kim, Jungtae; Woo, Dong-Cheol; Kim, Kyung Won; Song, Kyuyoung; Lee, Inchul

    2016-01-01

    Adipose tissue hyperplasia with increased number of adipocytes is implicated in a protective rather than deleterious effect on obesity-associated metabolic disorder. It is poorly understood how the adipose tissue cellularity is regulated. Tc1 is a gene of vertebrates that regulates diverse downstream genes. Young Tc1-deleted mice fed on standard chow diet show expanded adipose tissue with smaller adipocytes in size compared to wild type controls, representing adipose tissue hyperplasia. Tc1−/− mice show enhanced glucose tolerance and reduced serum lipids. Adipocyte-derived stem cells (ADSCs) from Tc1−/− mice show enhanced proliferative and adipogenic capacity compared to wild type controls, suggesting that the adipose hyperplasia is regulated at the stem cell level. PPARγ and CEBPα are up-regulated robustly in Tc1−/− ADSCs upon induction for adipogenesis. Wisp2 and Dlk1, inhibitors of adipogenesis, are down-regulated in Tc1−/− ADSCs compared to controls. Tc1-transfected NIH3T3 cells show higher β-catenin reporter signals than vector transfected controls, suggesting a role of canonical Wnt signaling in the Tc1-dependent adipose regulation. Our data support that Tc1 is a novel regulator for adipose stem cells. Adipose tissue hyperplasia may be implicated in the metabolic regulation of Tc1−/− mice. PMID:27775060

  18. Adipose tissue hyperplasia with enhanced adipocyte-derived stem cell activity in Tc1(C8orf4)-deleted mice.

    PubMed

    Jang, Hayoung; Kim, Minsung; Lee, Soyoung; Kim, Jungtae; Woo, Dong-Cheol; Kim, Kyung Won; Song, Kyuyoung; Lee, Inchul

    2016-10-24

    Adipose tissue hyperplasia with increased number of adipocytes is implicated in a protective rather than deleterious effect on obesity-associated metabolic disorder. It is poorly understood how the adipose tissue cellularity is regulated. Tc1 is a gene of vertebrates that regulates diverse downstream genes. Young Tc1-deleted mice fed on standard chow diet show expanded adipose tissue with smaller adipocytes in size compared to wild type controls, representing adipose tissue hyperplasia. Tc1(-/-) mice show enhanced glucose tolerance and reduced serum lipids. Adipocyte-derived stem cells (ADSCs) from Tc1(-/-) mice show enhanced proliferative and adipogenic capacity compared to wild type controls, suggesting that the adipose hyperplasia is regulated at the stem cell level. PPARγ and CEBPα are up-regulated robustly in Tc1(-/-) ADSCs upon induction for adipogenesis. Wisp2 and Dlk1, inhibitors of adipogenesis, are down-regulated in Tc1(-/-) ADSCs compared to controls. Tc1-transfected NIH3T3 cells show higher β-catenin reporter signals than vector transfected controls, suggesting a role of canonical Wnt signaling in the Tc1-dependent adipose regulation. Our data support that Tc1 is a novel regulator for adipose stem cells. Adipose tissue hyperplasia may be implicated in the metabolic regulation of Tc1(-/-) mice.

  19. Liver-specific deletion of Ppp2cα enhances glucose metabolism and insulin sensitivity.

    PubMed

    Xian, Li; Hou, Siyuan; Huang, Zan; Tang, An; Shi, Peiliang; Wang, Qinghua; Song, Anying; Jiang, Shujun; Lin, Zhaoyu; Guo, Shiying; Gao, Xiang

    2015-04-01

    Protein phosphatase 2A (PP2A) is a key negative regulator of phosphatidylinositol 3-kinase/Akt pathway. Previous study showed that, in the liver, the catalytic subunit of PP2A (PP2Ac) is closely associated with insulin resistance syndrome, which is characterized by glucose intolerance and dyslipidemia. Here we studied the role of liver PP2Ac in glucose metabolism and evaluated whether PP2Ac is a suitable therapeutic target for treating insulin resistance syndrome. Liver-specific Ppp2cα knockout mice (Ppp2cα(loxp/loxp): Alb) exhibited improved glucose homeostasis compared with littermate controls in both normal and high-fat diet conditions, despite no significant changes in body weight and liver weight under chow diet. Ppp2cα(loxp/loxp): Alb mice showed enhanced glycogen deposition, serum triglyceride, cholesterol, low density lipoprotein and high density lipoprotein, activated insulin signaling, decreased expressions of gluconeogenic genes G6P and PEPCK, and lower liver triglyceride. Liver-specific Ppp2cα knockout mice showed enhanced glucose homeostasis and increased insulin sensitivity by activation of insulin signaling through Akt. These findings suggest that inhibition of hepatic Ppp2cα may be a useful strategy for the treatment of insulin resistance syndrome.

  20. NAAG peptidase inhibitors and deletion of NAAG peptidase gene enhance memory in novel object recognition test

    PubMed Central

    Janczura, Karolina J.; Olszewski, Rafal T.; Bzdega, Tomasz; Bacich, Dean J.; Heston, Warren D.; Neale, Joseph H.

    2012-01-01

    The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is inactivated by the extracellular enzyme glutamate carboxypeptidase II. Inhibitors of this enzyme reverse dizocilpine (MK-801)-induced impairment of short-term memory in the novel object recognition test. The objective of this study was to test the hypothesis that NAAG peptidase inhibition enhances the long-term (24 hr delay) memory of C57BL mice in this test. These mice and mice in which glutamate carboxypeptidase II had been knocked out were presented with two identical objects to explore for 10 minutes on day 1 and tested with one of these familiar objects and one novel object on day 2. Memory was assessed as the degree to which the mice recalled the familiar object and explored the novel object to a greater extent on day 2. Uninjected mice or mice injected with saline prior to the acquisition session on day 1 demonstrated a lack of memory of the acquisition experience by exploring the familiar and novel objects to the same extent on day 2. Mice treated with glutamate carboxypeptidase II inhibitors ZJ43 or 2-PMPA prior to the acquisition trial explored the novel object significantly more time than the familiar object on day 2. Consistent with these results, mice in which glutamate carboxypeptidase II had been knocked out distinguished the novel from the familiar object on day 2 while their heterozygous colony mates did not. Inhibition of glutamate carboxypeptidase II enhances recognition memory, a therapeutic action that might be useful in treatment of memory deficits related to age and neurological disorders. PMID:23200894

  1. The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

    PubMed Central

    Sekelsky, J. J.; McKim, K. S.; Chin, G. M.; Hawley, R. S.

    1995-01-01

    Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion. PMID:8647398

  2. LKB1 deletion with the RIP2.Cre transgene modifies pancreatic β-cell morphology and enhances insulin secretion in vivo

    PubMed Central

    Sun, Gao; Tarasov, Andrei I.; McGinty, James A.; French, Paul M.; McDonald, Angela; Leclerc, Isabelle

    2010-01-01

    The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic β-cell and hypothalamic neurons using the rat insulin promoter (RIP2).Cre transgene (βAMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the β-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the β-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to βAMPKdKO mice, βLKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1-CreER mice, total β-cell mass and the size of individual islets and β-cells were increased and islet architecture was markedly altered in βLKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular Ca2+ were sharply reduced in βLKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on β-cell function. PMID:20354156

  3. The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilisation efficiency: implications for DNA repair.

    PubMed

    Toueille, Magali; El-Andaloussi, Nazim; Frouin, Isabelle; Freire, Raimundo; Funk, Dorothee; Shevelev, Igor; Friedrich-Heineken, Erica; Villani, Giuseppe; Hottiger, Michael O; Hübscher, Ulrich

    2004-01-01

    In eukaryotic cells, checkpoints are activated in response to DNA damage. This requires the action of DNA damage sensors such as the Rad family proteins. The three human proteins Rad9, Rad1 and Hus1 form a heterotrimeric complex (called the 9-1-1 complex) that is recruited onto DNA upon damage. DNA damage also triggers the recruitment of DNA repair proteins at the lesion, including specialized DNA polymerases. In this work, we showed that the 9-1-1 complex can physically interact with DNA polymerase beta in vitro. Functional analysis revealed that the 9-1-1 complex had a stimulatory effect on DNA polymerase beta activity. However, the presence of 9-1-1 complex neither affected DNA polymerase lambda, another X family DNA polymerase, nor the two replicative DNA polymerases alpha and delta. DNA polymerase beta stimulation resulted from an increase in its affinity for the primer-template and the interaction with the 9-1-1 complex stimulated deoxyribonucleotides misincorporation by DNA polymerase beta. In addition, the 9-1-1 complex enhanced DNA strand displacement synthesis by DNA polymerase beta on a 1 nt gap DNA substrate. Our data raise the possibility that the 9-1-1 complex might attract DNA polymerase beta to DNA damage sites, thus connecting directly checkpoints and DNA repair.

  4. Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

    PubMed

    Lee, Yeji; Nasution, Olviyani; Choi, Eunyong; Choi, In-Geol; Kim, Wankee; Choi, Wonja

    2015-08-01

    Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

  5. Deletion of Major Nonessential Genomic Regions in the Vaccinia Virus Lister Strain Enhances Attenuation without Altering Vaccine Efficacy in Mice▿

    PubMed Central

    Dimier, Julie; Ferrier-Rembert, Audrey; Pradeau-Aubreton, Karine; Hebben, Matthias; Spehner, Danièle; Favier, Anne-Laure; Gratier, Danielle; Garin, Daniel; Crance, Jean-Marc; Drillien, Robert

    2011-01-01

    The vaccinia virus (VACV) Lister strain was one of the vaccine strains that enabled smallpox eradication. Although the strain is most often harmless, there have been numerous incidents of mild to life-threatening accidents with this strain and others. In an attempt to further attenuate the Lister strain, we investigated the role of 5 genomic regions known to be deleted in the modified VACV Ankara (MVA) genome in virulence in immunodeficient mice, immunogenicity in immunocompetent mice, and vaccine efficacy in a cowpox virus challenge model. Lister mutants were constructed so as to delete each of the 5 regions or various combinations of these regions. All of the mutants replicated efficiently in tissue culture except region I mutants, which multiplied more poorly in human cells than the parental strain. Mutants with single deletions were not attenuated or only moderately so in athymic nude mice. Mutants with multiple deletions were more highly attenuated than those with single deletions. Deleting regions II, III, and V together resulted in total attenuation for nude mice and partial attenuation for SCID mice. In immunocompetent mice, the Lister deletion mutants induced VACV specific humoral responses equivalent to those of the parental strain but in some cases lower cell-mediated immune responses. All of the highly attenuated mutants protected mice from a severe cowpox virus challenge at low vaccine doses. The data suggest that several of the Lister mutants combining multiple deletions could be used in smallpox vaccination or as live virus vectors at doses equivalent to those used for the traditional vaccine while displaying increased safety. PMID:21367889

  6. Targeted deletion of the ara operon of Salmonella typhimurium enhances L-arabinose accumulation and drives PBAD-promoted expression of anti-cancer toxins and imaging agents.

    PubMed

    Hong, Hyun; Lim, Daejin; Kim, Geun-Joong; Park, Seung-Hwan; Sik Kim, Hyeon; Hong, Yeongjin; Choy, Hyon E; Min, Jung-Joon

    2014-01-01

    Tumor-specific expression of antitumor drugs can be achieved using attenuated Salmonella typhimurium harboring the PBAD promoter, which is induced by L-arabinose. However, L-arabinose does not accumulate because it is metabolized to D-xylulose-5-P by enzymes encoded by the ara operon in Salmonellae. To address this problem, we developed an engineered strain of S. typhimurium in which the ara operon is deleted. Linear DNA transformation was performed using λ red recombinase to exchange the ara operon with linear DNA carrying an antibiotic-resistance gene with homology to regions adjacent to the ara operon. The ara operon-deleted strain and its parental strain were transformed with a plasmid encoding Renilla luciferase variant 8 (RLuc8) or cytolysin A (clyA) under the control of the PBAD promoter. Luciferase assays demonstrated that RLuc8 expression was 49-fold higher in the ara operon-deleted S. typhimurium than in the parental strain after the addition of L-arabinose. In vivo bioluminescence imaging showed that the tumor tissue targeted by the ara operon-deleted Salmonella had a stronger imaging signal (~30-fold) than that targeted by the parental strain. Mice with murine colon cancer (CT26) that had been injected with the ara operon-deleted S. typhimurium expressing clyA showed significant tumor suppression. The present report demonstrates that deletion of the ara operon of S. typhimurium enhances L-arabinose accumulation and thereby drives PBAD-promoted expression of cytotoxic agents and imaging agents. This is a promising approach for tumor therapy and imaging.

  7. Conditional deletion of cardiomyocyte peroxisome proliferator-activated receptor γ enhances myocardial ischemia-reperfusion injury in mice.

    PubMed

    Hobson, Michael J; Hake, Paul W; O'Connor, Michael; Schulte, Christine; Moore, Victoria; James, Jeanne M; Piraino, Giovanna; Zingarelli, Basilia

    2014-01-01

    The nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of the inflammatory response to an array of biologic insults. We have previously demonstrated that PPARγ ligands reduce myocardial ischemia-reperfusion injury in rodents. In the current study, we directly determined the role of cardiomyocyte PPARγ in ischemia-reperfusion injury, using a model of conditional cardiomyocyte-specific deletion of PPARγ in vivo. In mice, α-myosin heavy chain-restricted Cre-mediated PPARγ deficiency was induced by tamoxifen treatment (30 mg/kg intraperitoneally) for 4 days (PPARγ mice), whereas controls included mice treated with the oil diluent vehicle (PPARγ mice). Western blot and histochemical analyses confirmed that expression of PPARγ protein was abolished in cardiomyocytes of mice treated with tamoxifen, but not with vehicle. After tamoxifen or vehicle treatment, animals were subjected to 30-min ligation of the left anterior descending coronary artery followed by 2-h reperfusion. In PPARγ mice, myocardial ischemia and reperfusion induced extensive myocardial damage, which was associated with elevated tissue activity of myeloperoxidase, indicating infiltration of neutrophils, and elevated plasma levels of troponin I when compared with PPARγ mice. Upon echocardiographic analysis, PPARγ mice also demonstrated ventricular dilatation and systolic dysfunction. Plasma levels of the proinflammatory cytokines interleukin 1β and interleukin 6 were higher in PPARγ mice when compared with PPARγ mice. These pathological events in PPARγ mice were associated with enhanced nuclear factor κB DNA binding in the infarcted hearts. Thus, our data suggest that cardiomyocyte PPARγ is a crucial protective receptor and may prevent reperfusion injury by modulating mechanisms of inflammation.

  8. In Vivo Deletion of the Cebpa +37 kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in Non-Hematopoietic Tissues to Impair Granulopoiesis

    PubMed Central

    Guo, Hong; Cooper, Stacy; Friedman, Alan D.

    2016-01-01

    The murine Cebpa gene contains a +37 kb, evolutionarily conserved 440 bp enhancer that directs high-level expression to myeloid progenitors in transgenic mice. The enhancer is bound and activated by Runx1, Scl, GATA2, C/EBPα, c-Myb, Pu.1, and additional Ets factors in myeloid cells. CRISPR/Cas9-mediated replacement of the wild-type enhancer with a variant mutant in its seven Ets sites leads to 20-fold reduction of Cebpa mRNA in the 32Dcl3 myeloid cell line. To determine the effect of deleting the enhancer in vivo, we now characterize C57BL/6 mice in which loxP sites flank a 688 bp DNA segment containing the enhancer. CMV-Cre mediated germline deletion resulted in diminution of the expected number of viable Enh(f/f);CMV-Cre offspring, with 28-fold reduction in marrow Cebpa mRNA but normal levels in liver, lung, adipose, intestine, muscle, and kidney. Cre-transduction of lineage-negative marrow cells in vitro reduced Cebpa mRNA 12-fold, with impairment of granulocytic maturation, morphologic blast accumulation, and IL-3 dependent myeloid colony replating for >12 generations. Exposure of Enh(f/f);Mx1-Cre mice to pIpC led to 14-fold reduction of Cebpa mRNA in GMP or CMP, 30-fold reduction in LSK, and <2-fold reduction in the LSK/SLAM subset. FACS analysis of marrow from these mice revealed 10-fold reduced neutrophils, 3-fold decreased GMP, and 3-fold increased LSK cells. Progenitor cell cycle progression was mildly impaired. Granulocyte and B lymphoid colony forming units were reduced while monocytic and erythroid colonies were increased, with reduced Pu.1 and Gfi1 and increased Egr1 and Klf4 in GMP. Finally, competitive transplantation indicated preservation of functional long-term hematopoietic stem cells upon enhancer deletion and confirmed marrow-intrinsic impairment of granulopoiesis and B cell generation with LSK and monocyte lineage expansion. These findings demonstrate a critical role for the +37 kb Cebpa enhancer for hematopoietic-specific Cebpa expression

  9. Deletion of glutamate delta-1 receptor in mouse leads to enhanced working memory and deficit in fear conditioning.

    PubMed

    Yadav, Roopali; Hillman, Brandon G; Gupta, Subhash C; Suryavanshi, Pratyush; Bhatt, Jay M; Pavuluri, Ratnamala; Stairs, Dustin J; Dravid, Shashank M

    2013-01-01

    Glutamate delta-1 (GluD1) receptors are expressed throughout the forebrain during development with high levels in the hippocampus during adulthood. We have recently shown that deletion of GluD1 receptor results in aberrant emotional and social behaviors such as hyperaggression and depression-like behaviors and social interaction deficits. Additionally, abnormal expression of synaptic proteins was observed in amygdala and prefrontal cortex of GluD1 knockout mice (GluD1 KO). However the role of GluD1 in learning and memory paradigms remains unknown. In the present study we evaluated GluD1 KO in learning and memory tests. In the eight-arm radial maze GluD1 KO mice committed fewer working memory errors compared to wildtype mice but had normal reference memory. Enhanced working memory in GluD1 KO was also evident by greater percent alternation in the spontaneous Y-maze test. No difference was observed in object recognition memory in the GluD1 KO mice. In the Morris water maze test GluD1 KO mice showed no difference in acquisition but had longer latency to find the platform in the reversal learning task. GluD1 KO mice showed a deficit in contextual and cue fear conditioning but had normal latent inhibition. The deficit in contextual fear conditioning was reversed by D-Cycloserine (DCS) treatment. GluD1 KO mice were also found to be more sensitive to foot-shock compared to wildtype. We further studied molecular changes in the hippocampus, where we found lower levels of GluA1, GluA2 and GluK2 subunits while a contrasting higher level of GluN2B in GluD1 KO. Additionally, we found higher postsynaptic density protein 95 (PSD95) and lower glutamate decarboxylase 67 (GAD67) expression in GluD1 KO. We propose that GluD1 is crucial for normal functioning of synapses and absence of GluD1 leads to specific abnormalities in learning and memory. These findings provide novel insights into the role of GluD1 receptors in the central nervous system.

  10. Deletion of Glutamate Delta-1 Receptor in Mouse Leads to Enhanced Working Memory and Deficit in Fear Conditioning

    PubMed Central

    Yadav, Roopali; Hillman, Brandon G.; Gupta, Subhash C.; Suryavanshi, Pratyush; Bhatt, Jay M.; Pavuluri, Ratnamala; Stairs, Dustin J.; Dravid, Shashank M.

    2013-01-01

    Glutamate delta-1 (GluD1) receptors are expressed throughout the forebrain during development with high levels in the hippocampus during adulthood. We have recently shown that deletion of GluD1 receptor results in aberrant emotional and social behaviors such as hyperaggression and depression-like behaviors and social interaction deficits. Additionally, abnormal expression of synaptic proteins was observed in amygdala and prefrontal cortex of GluD1 knockout mice (GluD1 KO). However the role of GluD1 in learning and memory paradigms remains unknown. In the present study we evaluated GluD1 KO in learning and memory tests. In the eight-arm radial maze GluD1 KO mice committed fewer working memory errors compared to wildtype mice but had normal reference memory. Enhanced working memory in GluD1 KO was also evident by greater percent alternation in the spontaneous Y-maze test. No difference was observed in object recognition memory in the GluD1 KO mice. In the Morris water maze test GluD1 KO mice showed no difference in acquisition but had longer latency to find the platform in the reversal learning task. GluD1 KO mice showed a deficit in contextual and cue fear conditioning but had normal latent inhibition. The deficit in contextual fear conditioning was reversed by D-Cycloserine (DCS) treatment. GluD1 KO mice were also found to be more sensitive to foot-shock compared to wildtype. We further studied molecular changes in the hippocampus, where we found lower levels of GluA1, GluA2 and GluK2 subunits while a contrasting higher level of GluN2B in GluD1 KO. Additionally, we found higher postsynaptic density protein 95 (PSD95) and lower glutamate decarboxylase 67 (GAD67) expression in GluD1 KO. We propose that GluD1 is crucial for normal functioning of synapses and absence of GluD1 leads to specific abnormalities in learning and memory. These findings provide novel insights into the role of GluD1 receptors in the central nervous system. PMID:23560106

  11. Deletion of an enhancer near DLX5 and DLX6 in a family with hearing loss, craniofacial defects, and an inv(7)(q21.3q35)

    PubMed Central

    Brown, Kerry K.; Reiss, Jacob A.; Crow, Kate; Ferguson, Heather L.; Kelly, Chantal; Fritzsch, Bernd; Morton, Cynthia C.

    2010-01-01

    Precisely regulated temporal and spatial patterns of gene expression are essential for proper human development. Cis-acting regulatory elements, some located at large distances from their corresponding genes, play a critical role in transcriptional control of key developmental genes and disruption of these regulatory elements can lead to disease. We report a three generation family with five affected members, all of whom have hearing loss, craniofacial defects, and a paracentric inversion of the long arm of chromosome 7, inv(7)(q21.3q35). High resolution mapping of the inversion showed that the 7q21.3 breakpoint is located 65 and 80 kb centromeric of DLX6 and DLX5, respectively. Further analysis revealed a 5115 bp deletion at the 7q21.3 breakpoint. While the breakpoint does not disrupt either DLX5 or DLX6, the syndrome present in the family is similar to that observed in Dlx5 knockout mice and includes a subset of the features observed in individuals with DLX5 and DLX6 deletions, implicating dysregulation of DLX5 and DLX6 in the family’s phenotype. Bioinformatic analysis indicates that the 5115 bp deletion at the 7q21.3 breakpoint could contain regulatory elements necessary for DLX5 and DLX6 expression. Using a transgenic mouse reporter assay, we show that the deleted sequence can drive expression in the ear and developing bones of E12.5 embryos. Consequently, the observed familial syndrome is likely caused by dysregulation of DLX5 and/or DLX6 in specific tissues due to deletion of an enhancer and possibly separation from other regulatory elements by the chromosomal inversion. PMID:19707792

  12. Deletion of atrophy enhancing genes fails to ameliorate the phenotype in a mouse model of spinal muscular atrophy.

    PubMed

    Iyer, Chitra C; McGovern, Vicki L; Wise, Dawnne O; Glass, David J; Burghes, Arthur H M

    2014-05-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disease causing degeneration of lower motor neurons and muscle atrophy. One therapeutic avenue for SMA is targeting signaling pathways in muscle to ameliorate atrophy. Muscle Atrophy F-box, MAFbx, and Muscle RING Finger 1, MuRF1, are muscle-specific ubiquitin ligases upregulated in skeletal and cardiac muscle during atrophy. Homozygous knock-out of MAFbx or MuRF1 causes muscle sparing in adult mice subjected to atrophy by denervation. We wished to determine whether blockage of the major muscle atrophy pathways by deletion of MAFbx or MuRF1 in a mouse model of SMA would improve the phenotype. Deletion of MAFbx in the Δ7 SMA mouse model had no effect on the weight and the survival of the mice while deletion of MuRF1 was deleterious. MAFbx(-/-)-SMA mice showed a significant alteration in fiber size distribution tending towards larger fibers. In skeletal and cardiac tissue MAFbx and MuRF1 transcripts were upregulated whereas MuRF2 and MuRF3 levels were unchanged in Δ7 SMA mice. We conclude that deletion of the muscle ubiquitin ligases does not improve the phenotype of a Δ7 SMA mouse. Furthermore, it seems unlikely that the beneficial effect of HDAC inhibitors is mediated through inhibition of MAFbx and MuRF1. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. An analysis of possible off target effects following CAS9/CRISPR targeted deletions of neuropeptide gene enhancers from the mouse genome.

    PubMed

    Hay, Elizabeth Anne; Khalaf, Abdulla Razak; Marini, Pietro; Brown, Andrew; Heath, Karyn; Sheppard, Darrin; MacKenzie, Alasdair

    2017-08-01

    We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome. However, reports of "off target" effects, whereby the CAS9 endonuclease is able to cut sites other than those targeted, limits the appeal of this technology. We used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes our analysis of the genomes of these enhancer knockout lines to detect possible off-target effects. Bioinformatic analysis was used to identify the most likely putative off-target sites and to design PCR primers that would amplify these sequences from genomic DNA of founder enhancer deletion mouse lines. Amplified DNA was then sequenced and blasted against the mouse genome sequence to detect off-target effects. Using this approach we were unable to detect any evidence of off-target effects in the genomes of three founder lines using any of the four gRNAs used in the analysis. This study suggests that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

    Cancer.gov

    The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

  15. Enhanced production of avermectin by deletion of type III polyketide synthases biosynthetic cluster rpp in Streptomyces avermitilis.

    PubMed

    Meng, L; Xiong, Z; Chu, J; Wang, Y

    2016-11-01

    The rpp biosynthetic gene cluster (sav7130-7131) in Streptomyces avermitilis contains a type III polyketide synthases (PKSs) and a cytochrome P450 and was reportedly involved in producing a diffusible brown pigment. Since the same precursor malonyl-CoA was used as substrate for the type I PKSs and type III PKSs, there might be a competition for precursor between rpp gene cluster and avermectin biosynthetic cluster. In this work, rpp biosynthetic gene cluster deletion mutants were constructed to improve avermectin production. In an industrial strain AV-LP, rpp deletion improved avermectin production from 1024 to 1262 mg l(-1) without any effect on the cell growth. In the same way, the production of an industrial overproducer increased from 3582 to 4450 mg l(-1) . Transcriptional analysis suggested that the deletion of rpp gene cluster stimulated transcription of aveR, leading to increased transcription of biosynthetic gene aveA1 and a consequent increase in avermectin production.

  16. Deletion of the Saccharomyces cerevisiae ARO8 gene, encoding an aromatic amino acid transaminase, enhances phenylethanol production from glucose.

    PubMed

    Romagnoli, Gabriele; Knijnenburg, Theo A; Liti, Gianni; Louis, Edward J; Pronk, Jack T; Daran, Jean-Marc

    2015-01-01

    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.

  17. Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine.

    PubMed

    Yin, Ying; Zhang, Sheng; Cai, Chenguang; Zhang, Jun; Dong, Dayong; Guo, Qiang; Fu, Ling; Xu, Junjie; Chen, Wei

    2014-02-01

    Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2β2-2β3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2β2-2β3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine. Copyright © 2013 Elsevier GmbH. All rights reserved.

  18. Combinatorial deletions of glgC and phaCE enhance ethanol production in Synechocystis sp. PCC 6803.

    PubMed

    Namakoshi, Katsunori; Nakajima, Tubasa; Yoshikawa, Katsunori; Toya, Yoshihiro; Shimizu, Hiroshi

    2016-12-10

    Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production. In the present study, a nitrogen starvation approach was applied on an ethanol producing strain for inhibiting the growth, since ethanol production competes with the cell growth. The effect of gene deletions in the glycogen and polyhydroxybutyrate (PHB) synthesis pathways was investigated. Measurements of intracellular glycogen and PHB revealed that the glycogen was accumulated under the nitrogen starvation condition and the gene deletion of glycogen synthesis pathway caused the accumulation of PHB. The ethanol producing strain harboring deletions for both the glycogen and the PHB synthesis pathways (ΔglgCΔphaCE/EtOH) produced ethanol at the specific rate of 240mgg (dry cell weight)(-1) day(-1) under the nitrogen starvation condition. In a high cell density culture (OD730=50) using this ΔglgCΔphaCE/EtOH strain, the ethanol production rates were 1.08 and 2.01gL(-1) day(-1) under light conditions of 40 and 80μmolm(-2)s(-1), respectively.

  19. Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

    PubMed Central

    Liao, Hua-Xin; Tomaras, Georgia D.; Bonsignori, Mattia; Tsao, Chun-Yen; Hwang, Kwan-Ki; Chen, Haiyan; Lloyd, Krissey E.; Bowman, Cindy; Sutherland, Laura; Jeffries, Thomas L.; Kozink, Daniel M.; Stewart, Shelley; Anasti, Kara; Jaeger, Frederick H.; Parks, Robert; Yates, Nicole L.; Overman, R. Glenn; Sinangil, Faruk; Berman, Phillip W.; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Karasavva, Nicos; Rerks-Ngarm, Supachai; Kim, Jerome H.; Michael, Nelson L.; Zolla-Pazner, Susan; Santra, Sampa; Letvin, Norman L.; Harrison, Stephen C.

    2013-01-01

    An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity. PMID:23175357

  20. A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD)

    DOE PAGES

    Giorgio, E.; Robyr, D.; Spielmann, M.; ...

    2015-02-20

    Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (~660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in amore » postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. Finally, this second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.« less

  1. A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD)

    SciTech Connect

    Giorgio, E.; Robyr, D.; Spielmann, M.; Ferrero, E.; Di Gregorio, E.; Imperiale, D.; Vaula, G.; Stamoulis, G.; Santoni, F.; Atzori, C.; Gasparini, L.; Ferrera, D.; Canale, C.; Guipponi, M.; Pennacchio, L. A.; Antonarakis, S. E.; Brussino, A.; Brusco, A.

    2015-02-20

    Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (~660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. Finally, this second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

  2. A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD).

    PubMed

    Giorgio, Elisa; Robyr, Daniel; Spielmann, Malte; Ferrero, Enza; Di Gregorio, Eleonora; Imperiale, Daniele; Vaula, Giovanna; Stamoulis, Georgios; Santoni, Federico; Atzori, Cristiana; Gasparini, Laura; Ferrera, Denise; Canale, Claudio; Guipponi, Michel; Pennacchio, Len A; Antonarakis, Stylianos E; Brussino, Alessandro; Brusco, Alfredo

    2015-06-01

    Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (∼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

  3. A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD)

    PubMed Central

    Giorgio, Elisa; Robyr, Daniel; Spielmann, Malte; Ferrero, Enza; Di Gregorio, Eleonora; Imperiale, Daniele; Vaula, Giovanna; Stamoulis, Georgios; Santoni, Federico; Atzori, Cristiana; Gasparini, Laura; Ferrera, Denise; Canale, Claudio; Guipponi, Michel; Pennacchio, Len A.; Antonarakis, Stylianos E.; Brussino, Alessandro; Brusco, Alfredo

    2015-01-01

    Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (∼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes. PMID:25701871

  4. Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains.

    PubMed

    Maiti, I B; Gowda, S; Kiernan, J; Ghosh, S K; Shepherd, R J

    1997-03-01

    The boundaries required for maximal expression from the promoter/leader region of the full length transcript of figwort mosaic virus (FLt promoter) coupled to reporter genes were defined by 5' and 3' deletion analyses. In transient expression assays using protoplasts of Nicotiana edwardsonii, a 314 bp FLt promoter fragment sequence (-249 to +65 from the transcription start site) was sufficient for strong expression activity. Plant expression vectors developed with modified FLt promoters were tested with GUS or CAT as reporter genes in transgenic plants. The FLt promoter is a strong constitutive promoter, with strength comparable to or greater than that of the CaMV 35S promoter. The FLt promoter with its double enhancer domain linked to GUS or CAT reporter genes provides an average 4-fold greater activity than the FLt promoter with a single enhancer domain (-55 to -249 bp upstream fragment) in tests with transgenic plants and in protoplast transient expression assays.

  5. Enhanced heterologous protein display on bacterial magnetic particles using a lon protease gene deletion mutant in Magnetospirillum magneticum AMB-1.

    PubMed

    Kanetsuki, Yuka; Tanaka, Tsuyoshi; Matsunaga, Tadashi; Yoshino, Tomoko

    2013-07-01

    Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1, are used as magnetic supports or carriers for a variety of biomedical and environmental applications. Although protein expression systems on BacMPs have been established in previous studies, the expression efficiency was dependent on the introduced protein sequences. Recombinant human proteins are often poorly expressed on BacMPs because of proteolytic degradation by endogenous proteases. We constructed a lon protease gene deletion mutant strain (Δlon) of M. magneticum AMB-1 by homologous recombination to increase the efficiency of functional protein display on BacMPs using Δlon host cells. Wild-type and Δlon-M. magneticum AMB-1 cells were transformed using expression plasmids for human proteins, thyroid-stimulating hormone receptor (TSHR) and the class II major histocompatibility complex (MHC II) molecules onto BacMPs. Although mRNA expression of both TSHR and MHC II was the same level in the wild-type and Δlon transformants, the protein expression levels in Δlon transformants were significantly increased versus wild-type cells. Furthermore, the amounts of two different human proteins on BacMPs were successfully improved. This phenomenon could be due to the reduction of the degradation of target proteins in the Δlon strain. This is the first report to construct a protease deletion mutant in magnetotactic bacteria. The Δlon strain is a useful host to provide BacMPs displaying target proteins for various experimental, and ultimately, clinical applications. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Long-term expression of von Willebrand Factor by a VSV-G pseudotyped lentivirus enhances the functional activity of secreted B-Domain-deleted Coagulation Factor VIII.

    PubMed

    Park, Sang Won; Choi, Sang-Yun

    2007-08-31

    von Willebrand factor (vWF) is a multimeric glycoprotein which functions within the coagulation system. It colocalizes with factor VIII (FVIII) by non-covalent interaction and alters its intracellular trafficking. vWF is also instrumental in maintaining the stability of secreted FVIII. The principal objective of this study was to generate a lentivirus-based vWF expression vector for gene therapy of hemophilia A. We inserted a vWF of 8.8 Kb into a lentiviral vector thereby producing VSV-G-pseudotyped vEx52. However, its titer was quite low, presumably because the length of vWF gene exceeds the size limit of the lentiviral vector. In order to overcome the low-titer, we concentrated the vEx52 and thus increased the efficiency of transduction approximately 6-fold with 1/100th of the volume. However, as concentration requires an additional laborious step, we attempted to enhance the transduction efficiency by deleting exons 24-46 and 29-46 in pRex52 to construct pRex23 and pRex28, and in pvEx52, yielding pvEx23 and pvEx28, respectively. The transfected pRex52 had a profound effect on the activity of secreted FVIII, and this activity declined as domains of vWF were deleted. However, when the domain-deleted vWF-lentiviruses were transduced into K562 cells, the vEx28 increased the activity of the secreted FVIII compared to what was observed with vEx52. This result is probably due to higher efficiencies of transduction and expression while retaining the essential domains required for proper interaction with FVIII.

  7. HBV polymerase overexpression due to large core gene deletion enhances hepatoma cell growth by binding inhibition of microRNA-100

    PubMed Central

    Huang, Ya-Hui; Tseng, Ying-Hsin; Lin, Wey-Ran; Hung, George; Chen, Tse-Ching; Wang, Tong-Hong; Lee, Wei-Chen; Yeh, Chau-Ting

    2016-01-01

    Different types of hepatitis B virus (HBV) core gene deletion mutants were identified in chronic hepatitis B patients. However, their clinical roles in different stages of natural chronic HBV infection remained unclear. To address this issue, HBV core genes were sequenced in three gender- and age-matched patient groups diagnosed as chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC), respectively. Functional analysis of the identified mutants was performed. A novel type of large-fragment core gene deletion (LFCD) was identified exclusively in HCC patients and significantly associated with unfavorable postoperative survival. The presence of LFCDs resulted in generation of precore-polymerase fusion protein or brought the polymerase reading frame under direct control of HBV precore/core promoter, leading to its over-expression. Enhanced cell proliferation and increased tumorigenicity in nude mice were found in hepatoma cells expressing LFCDs. Because of the epsilon-binding ability of HBV polymerase, we hypothesized that the over-expressed polymerase carrying aberrant amino-terminal sequence could bind to cellular microRNAs. Screening of a panel of microRNAs revealed physical association of a precore-polymerase fusion protein with microRNA-100. A binding inhibition effect on microRNA-100 by the precore-polymerase fusion protein with up-regulation of its target, polo-like kinase 1 (PLK1), was discovered. The binding inhibition and growth promoting effects could be reversed by overexpressing microRNA-100. Together, HCC patients carrying hepatitis B large-fragment core gene deletion mutants had an unfavorable postoperative prognosis. The growth promoting effect was partly due to polymerase overexpression, leading to binding inhibition of microRNA-100 and up-regulation of PLK1. PMID:26824500

  8. Enhanced freeze tolerance of baker's yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough.

    PubMed

    Tan, Haigang; Dong, Jian; Wang, Guanglu; Xu, Haiyan; Zhang, Cuiying; Xiao, Dongguang

    2014-08-01

    Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker's yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301(TPS1) overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301(TPS1) were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301(TPS1) was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker's yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker's yeast.

  9. Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato.

    PubMed

    Pan, I-Chun; Li, Chia-Wen; Su, Ruey-Chih; Cheng, Chiu-Ping; Lin, Choun-Sea; Chan, Ming-Tsair

    2010-10-01

    Ethylene-responsive transcription factors (ERFs) bind specifically to cis-acting DNA regulatory elements such as GCC boxes and play an important role in the regulation of defense- and stress-related genes in plants. In contrast to other ERFs, class II ERFs contain an ERF-associated amphiphilic repression (EAR) domain and act as GCC-mediated transcriptional repressors. In this study, SlERF3, a class II ERF was isolated from tomato and characterized. To examine whether the EAR motif of class II ERF proteins participates in ERF-mediated functions in plants, the EAR domain was deleted to generate SlERF3ΔRD. We show that SlERF3ΔRD protein retains the character of a transcription factor and becomes a GCC-mediated transcriptional activator. Constitutive expression of full-length SlERF3 in tomato severely suppressed growth and, as a result, no transgenic plants were obtained. However, no apparent effects on growth and development of SlERF3ΔRD transgenic plants were observed. Overexpression of SlERF3ΔRD in transgenic tomato induced expression of pathogenesis-related protein genes such as PR1, PR2 and PR5, and enhanced tolerance to Ralstonia solanacearum. Furthermore, transgenic Arabidopsis and tomatoes constitutively expressing SlERF3ΔRD exhibited reduced levels of membrane lipid peroxidation and enhanced tolerance to salt stress. In comparison with wild-type plants grown under stress conditions, transgenic SlERF3ΔRD tomatoes produced more flowers, fruits, and seeds. This study illustrates a gene-enhancing tolerance to both biotic and abiotic stresses in transgenic plants with the deletion of a repressor domain. Our findings suggest that class II ERF proteins may find important use in crop improvement or genetic engineering to increase stress tolerance in plants.

  10. Tip30 Deletion in MMTV-Neu Mice Leads to Enhanced EGFR Signaling and Development of Estrogen Receptor-Positive and Progesterone Receptor-Negative Mammary Tumors

    PubMed Central

    Zhang, Chengliang; Mori, Mikito; Gao, Shenglan; Li, Aimin; Hoshino, Isamu; Aupperlee, Mark D.; Haslam, Sandra Z.; Xiao, Hua

    2010-01-01

    Estrogen receptor-positive and progesterone receptor-negative (ER+/PR−) breast cancers account for 15–25% of all human breast cancers and display more aggressive malignant characteristics compared to ER+/PR+ cancers. However, the molecular mechanism underlying development of ER+/PR− breast cancers still remains elusive. We show here that Tip30 deletion dramatically accelerated the onset of mammary tumors in the MMTV-Neu mouse model of breast cancer. The mammary tumors arising in Tip30−/−/MMTV-Neu mice were exclusively ER+/PR−. The growth of these ER+/PR− tumors depends not only on estrogen but also on progesterone despite the absence of detectable PR. Tip30 is predominantly expressed in ER+ mammary epithelial cells (MECs) and its deletion leads to an increase in the number of phospho-ERα (p-ERα) positive cells in mammary glands and accelerated activation of Akt in MMTV-Neu mice. Moreover, we found that Tip30 regulates the EGFR pathway through controlling endocytic downregulation of EGFR protein level and signaling. Together, these findings suggest a novel mechanism in which loss of Tip30 cooperates with Neu activation to enhance the activation of Akt signaling, leading to the development of ER+/PR− mammary tumors. PMID:21159643

  11. Deletion of Galectin-3 Enhances Xenobiotic Induced Murine Primary Biliary Cholangitis by Facilitating Apoptosis of BECs and Release of Autoantigens.

    PubMed

    Arsenijevic, Aleksandar; Milovanovic, Marija; Milovanovic, Jelena; Stojanovic, Bojana; Zdravkovic, Natasa; Leung, Patrick S C; Liu, Fu-Tong; Gershwin, M Eric; Lukic, Miodrag L

    2016-03-21

    Galectin-3 (Gal-3) is a carbohydrate binding lectin, with multiple roles in inflammatory diseases and autoimmunity including its antiapoptotic effect on epithelial cells. In particular, increased expression of Gal-3 in epithelial cells is protective from apoptosis. Based on the thesis that apoptosis of biliary epithelial cells (BECs) is critical to the pathogenesis of Primary Biliary Cholangitis (PBC), we have analyzed the role of Gal-3 in the murine model of autoimmune cholangitis. We took advantage of Gal-3 knockout mice and immunized them with a mimotope of the major mitochondrial autoantigen of PBC, 2-octynoic acid (2-OA) coupled to BSA (2OA-BSA) and evaluated the natural history of subsequent disease, compared to control wild-type mice, by measuring levels of antibodies to PDC-E2, immunohistology of liver, and expression of Gal-3. We report herein that deletion of Gal-3 significantly exacerbates autoimmune cholangitis in these mice. This is manifested by increased periportal infiltrations, bile duct damage, granulomas and fibrosis. Interestingly, the BECs of Gal-3 knockout mice had a higher response to apoptotic stimuli and there were more pro-inflammatory lymphocytes and dendritic cells (DCs) in the livers of Gal-3 knockout mice. In conclusion, Gal-3 plays a protective role in the pathways that lead to the inflammatory destruction of biliary epithelial cells.

  12. MAL62 overexpression and NTH1 deletion enhance the freezing tolerance and fermentation capacity of the baker's yeast in lean dough.

    PubMed

    Sun, Xi; Zhang, Cui-Ying; Wu, Ming-Yue; Fan, Zhi-Hua; Liu, Shan-Na; Zhu, Wen-Bi; Xiao, Dong-Guang

    2016-04-04

    Trehalose is related to several types of stress responses, especially freezing response in baker's yeast (Saccharomyces cerevisiae). It is desirable to manipulate trehalose-related genes to create yeast strains that better tolerate freezing-thaw stress with improved fermentation capacity, which are in high demand in the baking industry. The strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention-freezing and long-term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing-thaw stress. The mutants of the industrial baker's yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.

  13. Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

    PubMed Central

    Leitner, Stephan; Sweeney, Katrina; Öberg, Daniel; Davies, Derek; Miranda, Enrique; Lemoine, Nick R.; Halldén, Gunnel

    2010-01-01

    Purpose Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal.We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways.These findings imply that less toxic doses than currently practicedin the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants. PMID:19223497

  14. Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability.

    PubMed

    Liu, H L; Doleyres, Y; Coutinho, P M; Ford, C; Reilly, P J

    2000-09-01

    Three single-residue mutations, Asp71-->Asn, Gln409-->Pro and Gly447-->Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28- Val29-Ser30-->Asn-Pro-Pro (23-30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301-->Ala-G ly-Ala (297-301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Delta439-441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N:-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O:-glycosylation in the already highly O:-glycosylated belt region that extends around the globular catalytic domain. The 23-30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297-301 replacement mutant GA was made to understand the function of the random coil region between alpha-helices 9 and 10. Delta439-441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23-30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4 degrees C increase in operating temperature at constant thermostability.

  15. bak deletion stimulates gastric epithelial proliferation and enhances Helicobacter felis-induced gastric atrophy and dysplasia in mice.

    PubMed

    Duckworth, C A; Abuderman, A A; Burkitt, M D; Williams, J M; O'Reilly, L A; Pritchard, D M

    2015-09-15

    Helicobacter infection causes a chronic superficial gastritis that in some cases progresses via atrophic gastritis to adenocarcinoma. Proapoptotic bak has been shown to regulate radiation-induced apoptosis in the stomach and colon and also susceptibility to colorectal carcinogenesis in vivo. Therefore we investigated the gastric mucosal pathology following H. felis infection in bak-null mice at 6 or 48 wk postinfection. Primary gastric gland culture from bak-null mice was also used to assess the effects of bak deletion on IFN-γ-, TNF-α-, or IL-1β-induced apoptosis. bak-null gastric corpus glands were longer, had increased epithelial Ki-67 expression, and contained fewer parietal and enteroendocrine cells compared with the wild type (wt). In wt mice, bak was expressed at the luminal surface of gastric corpus glands, and this increased 2 wk post-H. felis infection. Apoptotic cell numbers were decreased in bak-null corpus 6 and 48 wk following infection and in primary gland cultures following cytokine administration. Increased gastric epithelial Ki-67 labeling index was observed in C57BL/6 mice after H. felis infection, whereas no such increase was detected in bak-null mice. More severe gastric atrophy was observed in bak-null compared with C57BL/6 mice 6 and 48 wk postinfection, and 76% of bak-null compared with 25% of C57BL/6 mice showed evidence of gastric dysplasia following long-term infection. Collectively, bak therefore regulates gastric epithelial cell apoptosis, proliferation, differentiation, mucosal thickness, and susceptibility to gastric atrophy and dysplasia following H. felis infection. Copyright © 2015 the American Physiological Society.

  16. Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

    PubMed Central

    Milovanovic, Marija; Volarevic, Vladislav; Ljujic, Biljana; Radosavljevic, Gordana; Jovanovic, Ivan; Arsenijevic, Nebojsa; Lukic, Miodrag L.

    2012-01-01

    The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2−/− mice had significantly higher number of CD4+ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2−/− primed lymphocytes induced clinical signs of the disease in ST2−/− as well as in WT mice. MOG35–55 restimulated ST2−/− CD4+ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4+ cells. ST2−/− mice had higher percentages of CD4+ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4+ cells. Draining lymph nodes of ST2−/− mice contained higher percentage of CD11c+CD11b+CD8− cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2−/− but not WT dendritic cells induced EAE in MOG35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of

  17. C-terminal HIV-1 transframe p6* tetra-peptide blocks enhanced Gag cleavage incurred by leucine zipper replacement of a deleted p6* domain.

    PubMed

    Yu, Fu-Hsien; Huang, Kuo-Jung; Wang, Chin-Tien

    2017-03-01

    HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C-terminus). The data indicate that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetra-peptide plays a role in preventing premature PR maturation.IMPORTANCE Supporting evidence is lacking for the assumption that p6* retards PR maturation in the context of virus assembly. We found that replacing p6* with a leucine-zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (a) p6

  18. Studies on the O-specific polysaccharide of the lipopolysaccharide from the Pseudomonas mediterranea strain C5P1rad1, a bacterium pathogenic of tomato and chrysanthemum.

    PubMed

    Zdorovenko, Evelina L; Cimmino, Alessio; Marchi, Guido; Shashkov, Alexander S; Fiori, Mario; Knirel, Yuriy A; Evidente, Antonio

    2017-08-07

    An O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Pseudomonas mediterranea strain C5P1rad1, the causal agents of tomato pith necrosis and Chrysanthemum stem rot, and studied by one- and two-dimensional (1)H and (13)C NMR spectroscopy. The following structure of the trisaccharide repeating unit of the OPS was established, which, to our knowledge, is unique among the known bacterial polysaccharide structures: →4)-β-d-ManpNAc3NAcA-(1 → 4)-β-d-ManpNAc3NAcA-(1 → 3)-α-d-QuipNAc4NAc-(1→ where QuiNAc4NAc and ManNAc3NAcA indicate 2,4-diacetamido-2,4,6-trideoxyglucose and 2,3-diacetamido-2,3-dideoxymannuronic acid, respectively. Pre-treatment of leaves with LPS or OPS preparations at 250 and 50 μg mL(-1) did not inhibit development of a hypersensitivity reaction induced by P. mediterranea C5P1rad1 on tobacco, tomato and chrysanthemum plants. The same preparations at 250 μg mL(-1) partially prevented elicitation of the hypersensitivity reaction by Pseudomonas syringae KVPT7RC on chrysanthemum but not tobacco and tomato. Copyright © 2017. Published by Elsevier Ltd.

  19. PTEN deletion enhances survival, neurite outgrowth and function of dopamine neuron grafts to MitoPark mice

    PubMed Central

    Zhang, YaJun; Granholm, Ann-Charlotte; Huh, Kyounghee; Shan, Lufei; Diaz-Ruiz, Oscar; Malik, Nasir; Olson, Lars; Hoffer, Barry J.; Lupica, Carl R.; Hoffman, Alexander F.

    2012-01-01

    Clinical trials in Parkinson’s disease have shown that transplants of embryonic mesencephalic dopamine neurons form new functional connections within the host striatum, but the therapeutic benefits have been highly variable. One obstacle has been poor survival and integration of grafted dopamine neurons. Activation of Akt, a serine/threonine kinase that promotes cell survival and growth, increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. Because the lipid phosphatase, phosphatase and tensin homolog (PTEN) inhibits Akt, we generated a mouse with conditional knock-out of PTEN in dopamine neurons, leading to constitutive expression of Akt in these neurons. Ventral mesencephalic tissue from dopamine phosphatase and tensin homologue knock-out or control animals was then transplanted bilaterally into the dopamine depleted striata of MitoPark mice that express a parkinsonian phenotype because of severe respiratory chain dysfunction in dopamine neurons. After transplantation into MitoPark mice, PTEN-deficient dopamine neurons were less susceptible to cell death, and exhibited a more extensive pattern of fibre outgrowth compared to control grafts. Voltammetric measurements demonstrated that dopamine release and reuptake were significantly increased in the striata of animals receiving dopamine PTEN knock-out transplants. These animals also displayed enhanced spontaneous and drug-induced locomotor activity, relative to control transplanted MitoPark mice. Our results suggest that disinhibition of the Akt-signalling pathway may provide a valuable strategy to enhance survival, function and integration of grafted dopamine neurons within the host striatum and, more generally, to improve survival and integration of different forms of neural grafts. PMID:22961549

  20. Enhanced production of branched-chain amino acids by Gluconacetobacter europaeus with a specific regional deletion in a leucine responsive regulator.

    PubMed

    Akasaka, Naoki; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

    2014-12-01

    Vinegar with increased amounts of branched-chain amino acids (BCAAs; valine, leucine and isoleucine) is favorable for human health as BCAAs decrease diet-induced obesity and hyperglycemia. To construct Gluconacetobacter europaeus which produces BCAAs, leucine responsive regulator (GeLrp) is focused and two Gelrp mutants were constructed. Wild-type KGMA0119 didn't produce significant amount of valine (0.13 mM) and leucine (0 mM) and strain KGMA7110 which lacks complete Gelrp accumulated valine (0.48 mM) and leucine (0.11 mM) but showed impaired growth, and it was fully restored in the presence of essential amino acids. Strain KGMA7203 was then constructed with a nonsense mutation at codon Trp132 in the Gelrp, which leads a specific deletion at an estimated ligand-sensing region in the C-terminal domain. KGMA7203 produced greater quantities of valine (0.80 mM) and leucine (0.26 mM) and showed the same growth characteristics as KGMA0119. mRNA levels of BCAAs biosynthesis genes (ilvI and ilvC) and probable BCAAs efflux pump (leuE) were determined by quantitative reverse-transcription PCR. Expression rates of ilvI and ilvC in the two Gelrp disruptants were greater than those in KGMA0119. leuE was highly expressed in KGMA7110 only, suggesting that the accumulation in KGMA7110 culture was caused by increased expression of the biosynthesis genes and abnormal enhanced export of amino acids resulting in impaired cell growth. In contrast, KGMA7203 would achieve the high level production through enhanced expression of the biosynthesis genes without enhancing that for the efflux pump. KGMA7203 was considered advantageous for production of vinegar with higher amounts of valine and leucine.

  1. Deletion of A44L, A46R and C12L Vaccinia Virus Genes from the MVA Genome Improved the Vector Immunogenicity by Modifying the Innate Immune Response Generating Enhanced and Optimized Specific T-Cell Responses.

    PubMed

    Holgado, María Pía; Falivene, Juliana; Maeto, Cynthia; Amigo, Micaela; Pascutti, María Fernanda; Vecchione, María Belén; Bruttomesso, Andrea; Calamante, Gabriela; Del Médico-Zajac, María Paula; Gherardi, María Magdalena

    2016-05-23

    MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R); or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R). The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/b⁺/IFN-γ⁺) and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1β and IFN-β. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential.

  2. Deletion of A44L, A46R and C12L Vaccinia Virus Genes from the MVA Genome Improved the Vector Immunogenicity by Modifying the Innate Immune Response Generating Enhanced and Optimized Specific T-Cell Responses

    PubMed Central

    Holgado, María Pía; Falivene, Juliana; Maeto, Cynthia; Amigo, Micaela; Pascutti, María Fernanda; Vecchione, María Belén; Bruttomesso, Andrea; Calamante, Gabriela; del Médico-Zajac, María Paula; Gherardi, María Magdalena

    2016-01-01

    MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R); or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R). The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/b+/IFN-γ+) and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1β and IFN-β. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential. PMID:27223301

  3. An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression.

    PubMed

    Wyszynski, Asaf; Hong, Chi-Chen; Lam, Kristin; Michailidou, Kyriaki; Lytle, Christian; Yao, Song; Zhang, Yali; Bolla, Manjeet K; Wang, Qin; Dennis, Joe; Hopper, John L; Southey, Melissa C; Schmidt, Marjanka K; Broeks, Annegien; Muir, Kenneth; Lophatananon, Artitaya; Fasching, Peter A; Beckmann, Matthias W; Peto, Julian; Dos-Santos-Silva, Isabel; Sawyer, Elinor J; Tomlinson, Ian; Burwinkel, Barbara; Marme, Frederik; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E; Nordestgaard, Børge G; González-Neira, Anna; Benitez, Javier; Neuhausen, Susan L; Brenner, Hermann; Dieffenbach, Aida Karina; Meindl, Alfons; Schmutzler, Rita K; Brauch, Hiltrud; Nevanlinna, Heli; Khan, Sofia; Matsuo, Keitaro; Ito, Hidemi; Dörk, Thilo; Bogdanova, Natalia V; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kosma, Veli-Matti; Wu, Anna H; Van Den Berg, David; Lambrechts, Diether; Wildiers, Hans; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Peterlongo, Paolo; Couch, Fergus J; Olson, Janet E; Giles, Graham G; Milne, Roger L; Haiman, Christopher A; Henderson, Brian E; Dumont, Martine; Teo, Soo Hwang; Wong, Tien Y; Kristensen, Vessela; Zheng, Wei; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Andrulis, Irene L; Knight, Julia A; Devilee, Peter; Seynaeve, Caroline; García-Closas, Montserrat; Figueroa, Jonine; Klevebring, Daniel; Czene, Kamila; Hooning, Maartje J; van den Ouweland, Ans M W; Darabi, Hatef; Shu, Xiao-Ou; Gao, Yu-Tang; Cox, Angela; Blot, William; Signorello, Lisa B; Shah, Mitul; Kang, Daehee; Choi, Ji-Yeob; Hartman, Mikael; Miao, Hui; Hamann, Ute; Jakubowska, Anna; Lubinski, Jan; Sangrajrang, Suleeporn; McKay, James; Toland, Amanda E; Yannoukakos, Drakoulis; Shen, Chen-Yang; Wu, Pei-Ei; Swerdlow, Anthony; Orr, Nick; Simard, Jacques; Pharoah, Paul D P; Dunning, Alison M; Chenevix-Trench, Georgia; Hall, Per; Bandera, Elisa; Amos, Chris; Ambrosone, Christine; Easton, Douglas F; Cole, Michael D

    2016-09-01

    Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following oestrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR = 0.68 95%CI 0.55-0.83, P = 0.0002; replication OR = 0.77 95% CI 0.73-0.82, P = 2.1 × 10(-19)) and identify 13 additional linked variants (r(2 )>( )0.8) in the 20Kb linkage block containing the enCNV (P = 3.2 × 10(-15) - 5.6 × 10(-17)). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5.

  4. An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression

    PubMed Central

    Wyszynski, Asaf; Hong, Chi-Chen; Lam, Kristin; Michailidou, Kyriaki; Lytle, Christian; Yao, Song; Zhang, Yali; Bolla, Manjeet K.; Wang, Qin; Dennis, Joe; Hopper, John L.; Southey, Melissa C.; Schmidt, Marjanka K.; Broeks, Annegien; Muir, Kenneth; Lophatananon, Artitaya; Fasching, Peter A.; Beckmann, Matthias W.; Peto, Julian; dos-Santos-Silva, Isabel; Sawyer, Elinor J.; Tomlinson, Ian; Burwinkel, Barbara; Marme, Frederik; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E.; Nordestgaard, Børge G.; González-Neira, Anna; Benitez, Javier; Neuhausen, Susan L.; Brenner, Hermann; Dieffenbach, Aida Karina; Meindl, Alfons; Schmutzler, Rita K.; Brauch, Hiltrud; Nevanlinna, Heli; Khan, Sofia; Matsuo, Keitaro; Ito, Hidemi; Dörk, Thilo; Bogdanova, Natalia V.; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kosma, Veli-Matti; Wu, Anna H.; Van Den Berg, David; Lambrechts, Diether; Wildiers, Hans; Chang-Claude, Jenny; Rudolph, Anja; Radice, Paolo; Peterlongo, Paolo; Couch, Fergus J.; Olson, Janet E.; Giles, Graham G.; Milne, Roger L.; Haiman, Christopher A.; Henderson, Brian E.; Dumont, Martine; Teo, Soo Hwang; Wong, Tien Y.; Kristensen, Vessela; Zheng, Wei; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Andrulis, Irene L.; Knight, Julia A.; Devilee, Peter; Seynaeve, Caroline; García-Closas, Montserrat; Figueroa, Jonine; Klevebring, Daniel; Czene, Kamila; Hooning, Maartje J.; van den Ouweland, Ans M.W.; Darabi, Hatef; Shu, Xiao-Ou; Gao, Yu-Tang; Cox, Angela; Blot, William; Signorello, Lisa B.; Shah, Mitul; Kang, Daehee; Choi, Ji-Yeob; Hartman, Mikael; Miao, Hui; Hamann, Ute; Jakubowska, Anna; Lubinski, Jan; Sangrajrang, Suleeporn; McKay, James; Toland, Amanda E.; Yannoukakos, Drakoulis; Shen, Chen-Yang; Wu, Pei-Ei; Swerdlow, Anthony; Orr, Nick; Simard, Jacques; Pharoah, Paul D.P.; Dunning, Alison M.; Chenevix-Trench, Georgia; Hall, Per; Bandera, Elisa; Amos, Chris; Ambrosone, Christine; Easton, Douglas F.; Cole, Michael D.

    2016-01-01

    Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following oestrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR = 0.68 95%CI 0.55–0.83, P = 0.0002; replication OR = 0.77 95% CI 0.73-0.82, P = 2.1 × 10−19) and identify 13 additional linked variants (r2 > 0.8) in the 20Kb linkage block containing the enCNV (P = 3.2 × 10−15 − 5.6 × 10−17). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5. PMID:27402876

  5. Deletion mutation of sodium channel Na(V)1.7 in inherited erythromelalgia: enhanced slow inactivation modulates dorsal root ganglion neuron hyperexcitability.

    PubMed

    Cheng, Xiaoyang; Dib-Hajj, Sulayman D; Tyrrell, Lynda; Te Morsche, Rene H; Drenth, Joost P H; Waxman, Stephen G

    2011-07-01

    Gain-of-function missense mutations of voltage-gated sodium channel Na(V)1.7 have been linked to the painful disorder inherited erythromelalgia. These mutations hyperpolarize activation, slow deactivation and enhance currents evoked by slow ramp stimuli (ramp currents). A correlation has recently been suggested between the age of onset of inherited erythromelalgia and the extent of hyperpolarizing shifts in mutant Na(V)1.7 channel activation; mutations causing large activation shifts have been linked to early age of onset inherited erythromelalgia, while mutations causing small activation shifts have been linked to age of onset within the second decade of life. Here, we report a family with inherited erythromelalgia with an in-frame deletion of a single residue--leucine 955 (Del-L955) in DII/S6. The proband did not show symptoms until the age of 15 years, and her affected mother only experienced mild symptoms during adolescence, which disappeared at the age of 38 years. Del-L955 shows no effect on Na(V)1.7 current density and fast inactivation, but causes an approximately -24 mV shift in activation, together with increases in amplitude of persistent currents and ramp currents. The mutation also produces an approximately -40 mV shift in slow inactivation, which reduces channel availability. Comparison of the effects of the Del-L955 mutation on dorsal root ganglion neuron hyperexcitability with those produced by another inherited erythromelalgia mutation (L858F) that does not enhance slow inactivation suggests that a delayed age of onset and milder symptoms in association with a large shift of channel activation, enhanced persistent and enhanced ramp currents may be related to the approximately -40 mV shift in slow inactivation for Del-L955, the largest shift thus far demonstrated in mutant Na(V)1.7 channels. Our results suggest that despite the pivotal role of activation shift in inherited erythromelalgia development, slow inactivation may regulate clinical

  6. Deletion of calcineurin and myocyte enhancer factor 2 (MEF2) binding domain of Cabin1 results in enhanced cytokine gene expression in T cells.

    PubMed

    Esau, C; Boes, M; Youn, H D; Tatterson, L; Liu, J O; Chen, J

    2001-11-19

    Cabin1 binds calcineurin and myocyte enhancer factor 2 (MEF2) through its COOH-terminal region. In cell lines, these interactions were shown to inhibit calcineurin activity after T cell receptor (TCR) signaling and transcriptional activation of Nur77 by MEF2. The role of these interactions under physiological conditions was investigated using a mutant mouse strain that expresses a truncated Cabin1 lacking the COOH-terminal calcineurin and MEF2 binding domains. T and B cell development and thymocyte apoptosis were normal in mutant mice. In response to anti-CD3 stimulation, however, mutant T cells expressed significantly higher levels of interleukin (IL)-2, IL-4, IL-9, IL-13, and interferon gamma than wild-type T cells. The enhanced cytokine gene expression was not associated with change in nuclear factor of activated T cells (NF-AT)c or NF-ATp nuclear translocation but was preceded by the induction of a phosphorylated form of MEF2D in mutant T cells. Consistent with the enhanced cytokine expression, mutant mice had elevated levels of serum immunoglobulin (Ig)G1, IgG2b, and IgE and produced more IgG1 in response to a T cell-dependent antigen. These findings suggest that the calcineurin and MEF2 binding domain of Cabin1 is dispensable for thymocyte development and apoptosis, but is required for proper regulation of T cell cytokine expression probably through modulation of MEF2 activity.

  7. Genetic Deletion of Neuronal PPARγ Enhances the Emotional Response to Acute Stress and Exacerbates Anxiety: An Effect Reversed by Rescue of Amygdala PPARγ Function.

    PubMed

    Domi, Esi; Uhrig, Stefanie; Soverchia, Laura; Spanagel, Rainer; Hansson, Anita C; Barbier, Estelle; Heilig, Markus; Ciccocioppo, Roberto; Ubaldi, Massimo

    2016-12-14

    attracted attention for its involvement in the regulation of CNS immune response and functions. Here, we demonstrate that neuronal PPARγ activation prevented the negative emotional effects of stress and exerted anxiolytic actions without influencing hypothalamic-pituitary-adrenal axis function. Conversely, pharmacological blockade or genetic deletion of PPARγ enhanced anxiogenic responses and increased vulnerability to stress. These effects appear to be controlled by PPARγ neuronal-mediated mechanisms in the amygdala. Copyright © 2016 the authors 0270-6474/16/3612612-13$15.00/0.

  8. Interaction between Rad9-Hus1-Rad1 and TopBP1 activates ATR-ATRIP and promotes TopBP1 recruitment to sites of UV-damage.

    PubMed

    Ohashi, Eiji; Takeishi, Yukimasa; Ueda, Satoshi; Tsurimoto, Toshiki

    2014-09-01

    The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9-1-1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9-1-1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Evolutionary conservation analysis increases the colocalization of predicted exonic splicing enhancers in the BRCA1 gene with missense sequence changes and in-frame deletions, but not polymorphisms

    PubMed Central

    Pettigrew, Christopher; Wayte, Nicola; Lovelock, Paul K; Tavtigian, Sean V; Chenevix-Trench, Georgia; Spurdle, Amanda B; Brown, Melissa A

    2005-01-01

    Introduction Aberrant pre-mRNA splicing can be more detrimental to the function of a gene than changes in the length or nature of the encoded amino acid sequence. Although predicting the effects of changes in consensus 5' and 3' splice sites near intron:exon boundaries is relatively straightforward, predicting the possible effects of changes in exonic splicing enhancers (ESEs) remains a challenge. Methods As an initial step toward determining which ESEs predicted by the web-based tool ESEfinder in the breast cancer susceptibility gene BRCA1 are likely to be functional, we have determined their evolutionary conservation and compared their location with known BRCA1 sequence variants. Results Using the default settings of ESEfinder, we initially detected 669 potential ESEs in the coding region of the BRCA1 gene. Increasing the threshold score reduced the total number to 464, while taking into consideration the proximity to splice donor and acceptor sites reduced the number to 211. Approximately 11% of these ESEs (23/211) either are identical at the nucleotide level in human, primates, mouse, cow, dog and opossum Brca1 (conserved) or are detectable by ESEfinder in the same position in the Brca1 sequence (shared). The frequency of conserved and shared predicted ESEs between human and mouse is higher in BRCA1 exons (2.8 per 100 nucleotides) than in introns (0.6 per 100 nucleotides). Of conserved or shared putative ESEs, 61% (14/23) were predicted to be affected by sequence variants reported in the Breast Cancer Information Core database. Applying the filters described above increased the colocalization of predicted ESEs with missense changes, in-frame deletions and unclassified variants predicted to be deleterious to protein function, whereas they decreased the colocalization with known polymorphisms or unclassified variants predicted to be neutral. Conclusion In this report we show that evolutionary conservation analysis may be used to improve the specificity of an ESE

  10. Deletion of meso-2,3-butanediol dehydrogenase gene budC for enhanced D-2,3-butanediol production in Bacillus licheniformis

    PubMed Central

    2014-01-01

    Background D-2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional D-2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogenic and not capable of producing D-2,3-butanediol at high optical purity. Bacillus licheniformis is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of D- and meso-type isomers. BudC in B. licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis. Results We developed a genetically modified strain of B. licheniformis (WX-02 ΔbudC) as a D-2,3-butanediol producer with high optimal purity. A marker-less gene deletion protocol based on a temperature sensitive knock-out plasmid T2-Ori was used to knock out the budC gene in B. licheniformis WX-02. The budC knock-out strain successfully abolished meso-2,3-butanediol production with enhanced D-2,3-butanediol production. No meso-BDH activity was detectable in cells of this strain. On the other hand, the complementary strain restored the characteristics of wild strain, and produced meso-2,3-butanediol and possessed meso-BDH activity. All of these data suggested that budC encoded the major meso-BDH catalyzing the reversible reaction from acetoin to meso-2,3-butanediol in B. licheniformis. The budC knock-out strain produced D-2,3-butanediol isomer only with a high yield of 30.76 g/L and a productivity of 1.28 g/L-h. Conclusions We confirmed the hypothesis that budC gene is responsible to reversibly transfer acetoin to meso-2,3-butanediol in B. licheniformis. A mutant strain of B. licheniformis with depleted budC gene was successfully developed and produced high level of the D-2,3-butanediol with high optimal purity. PMID:24475980

  11. Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C

    PubMed Central

    Perdiguero, Beatriz; Gómez, Carmen Elena; Di Pilato, Mauro; Sorzano, Carlos Oscar S.; Delaloye, Julie; Roger, Thierry; Calandra, Thierry; Pantaleo, Giuseppe; Esteban, Mariano

    2013-01-01

    Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates. PMID:24069354

  12. Interleukin-1- and Type I Interferon-Dependent Enhanced Immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef Vaccine Vector with Dual Deletions of Type I and Type II Interferon-Binding Proteins

    PubMed Central

    Delaloye, Julie; Filali-Mouhim, Abdelali; Cameron, Mark J.; Haddad, Elias K.; Harari, Alexandre; Goulet, Jean-Pierre; Gomez, Carmen E.; Perdiguero, Beatriz; Esteban, Mariano; Pantaleo, Giuseppe; Roger, Thierry; Sékaly, Rafick-Pierre

    2015-01-01

    ABSTRACT NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4+ T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV

  13. Interleukin-1- and type I interferon-dependent enhanced immunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nef vaccine vector with dual deletions of type I and type II interferon-binding proteins.

    PubMed

    Delaloye, Julie; Filali-Mouhim, Abdelali; Cameron, Mark J; Haddad, Elias K; Harari, Alexandre; Goulet, Jean-Pierre; Gomez, Carmen E; Perdiguero, Beatriz; Esteban, Mariano; Pantaleo, Giuseppe; Roger, Thierry; Sékaly, Rafick-Pierre; Calandra, Thierry

    2015-04-01

    NYVAC, a highly attenuated, replication-restricted poxvirus, is a safe and immunogenic vaccine vector. Deletion of immune evasion genes from the poxvirus genome is an attractive strategy for improving the immunogenic properties of poxviruses. Using systems biology approaches, we describe herein the enhanced immunological profile of NYVAC vectors expressing the HIV-1 clade C env, gag, pol, and nef genes (NYVAC-C) with single or double deletions of genes encoding type I (ΔB19R) or type II (ΔB8R) interferon (IFN)-binding proteins. Transcriptomic analyses of human monocytes infected with NYVAC-C, NYVAC-C with the B19R deletion (NYVAC-C-ΔB19R), or NYVAC-C with B8R and B19R deletions (NYVAC-C-ΔB8RB19R) revealed a concerted upregulation of innate immune pathways (IFN-stimulated genes [ISGs]) of increasing magnitude with NYVAC-C-ΔB19R and NYVAC-C-ΔB8RB19R than with NYVAC-C. Deletion of B8R and B19R resulted in an enhanced activation of IRF3, IRF7, and STAT1 and the robust production of type I IFNs and of ISGs, whose expression was inhibited by anti-type I IFN antibodies. Interestingly, NYVAC-C-ΔB8RB19R induced the production of much higher levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-6 [IL-6], and IL-8) than NYVAC-C or NYVAC-C-ΔB19R as well as a strong inflammasome response (caspase-1 and IL-1β) in infected monocytes. Top network analyses showed that this broad response mediated by the deletion of B8R and B19R was organized around two upregulated gene expression nodes (TNF and IRF7). Consistent with these findings, monocytes infected with NYVAC-C-ΔB8RB19R induced a stronger type I IFN-dependent and IL-1-dependent allogeneic CD4(+) T cell response than monocytes infected with NYVAC-C or NYVAC-C-ΔB19R. Dual deletion of type I and type II IFN immune evasion genes in NYVAC markedly enhanced its immunogenic properties via its induction of the increased expression of type I IFNs and IL-1β and make it an attractive candidate HIV

  14. Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice.

    PubMed

    Luckow, Bruno; Hänggli, Amy; Maier, Holger; Chilla, Silvia; Loewe, Robert P; Dehmel, Stefan; Schlöndorff, Detlef; Loetscher, Pius; Zerwes, Hans-Günter; Müller, Matthias

    2009-08-01

    The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.

  15. A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9-Rad1-Hus1 Checkpoint Complex Interactions

    SciTech Connect

    P Luncsford; D Chang; G Shi; J Bernstein; A Madabushi; D Patterson; A Lu; E Toth

    2011-12-31

    The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

  16. A structural hinge in eukaryotic MutY homologues mediates catalytic activity and Rad9-Rad1-Hus1 checkpoint complex interactions.

    PubMed

    Luncsford, Paz J; Chang, Dau-Yin; Shi, Guoli; Bernstein, Jade; Madabushi, Amrita; Patterson, Dimeka N; Lu, A-Lien; Toth, Eric A

    2010-10-29

    The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

  17. Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics.

    PubMed

    Peacock, Thomas P; Benton, Donald J; James, Joe; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; Barclay, Wendy S; Iqbal, Munir

    2017-07-15

    H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence.IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are able to

  18. Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics

    PubMed Central

    Peacock, Thomas P.; Benton, Donald J.; James, Joe; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E.; Bryant, Juliet E.; Martin, Stephen R.; Barclay, Wendy S.

    2017-01-01

    ABSTRACT H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence. IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are

  19. Deletion of Stat3 enhances myeloid cell expansion and increases the severity of myeloproliferative neoplasms in Jak2V617F knock-in mice

    PubMed Central

    Yan, Dongqing; Jobe, Fatoumata; Hutchison, Robert E.; Mohi, Golam

    2015-01-01

    The JAK2V617F mutation commonly found in myeloproliferative neoplasms (MPNs) induces constitutive phosphorylation/activation of the signal transducer and activator of transcription 3 (Stat3). However, the contribution of Stat3 in MPN evoked by JAK2V617F remains unknown. To determine the role of Stat3 in JAK2V617F-induced MPN, we generated Stat3-deficient Jak2V617F-expressing mice. Whereas expression of Jak2V617F resulted in a PV-like disease characterized by increased red blood cells (RBC), hematocrit, neutrophils and platelets in the peripheral blood of Jak2V617F knock-in mice, deletion of Stat3 slightly reduced RBC, and hematocrit parameters and modestly increased platelet numbers in Jak2V617F knock-in mice. Moreover, deletion of Stat3 significantly increased the neutrophil counts/percentages and markedly reduced the survival of mice expressing Jak2V617F. These phenotypic manifestations were reproduced upon bone marrow transplantation into wild-type animals. Flow cytometric analysis showed increased hematopoietic stem cell and granulocyte-macrophage progenitor populations in the bone marrow and spleens of Stat3-deficient Jak2V617F mice. Stat3 deficiency also caused a marked expansion of Gr-1+/Mac-1+ myeloid cells in Jak2V617F knock-in mice. Histopathologic analysis revealed marked increase in granulocytes in the bone marrow, spleens and livers of Stat3-deficient Jak2V617F-expressing mice. Together, these results suggest that deletion of Stat3 increases the severity of MPN induced by Jak2V617F. PMID:26044284

  20. Schizophrenia and chromosomal deletions

    SciTech Connect

    Lindsay, E.A.; Baldini, A.; Morris, M. A.

    1995-06-01

    Recent genetic linkage analysis studies have suggested the presence of a schizophrenia locus on the chromosomal region 22q11-q13. Schizophrenia has also been frequently observed in patients affected with velo-cardio-facial syndrome (VCFS), a disorder frequently associated with deletions within 22q11.1. It has been hypothesized that psychosis in VCFS may be due to deletion of the catechol-o-methyl transferase gene. Prompted by these observations, we screened for 22q11 deletions in a population of 100 schizophrenics selected from the Maryland Epidemiological Sample. Our results show that there are schizophrenic patients carrying a deletion of 22q11.1 and a mild VCFS phenotype that might remain unrecognized. These findings should encourage a search for a schizophrenia-susceptibility gene within the deleted region and alert those in clinical practice to the possible presence of a mild VCFS phenotype associated with schizophrenia. 9 refs.

  1. Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes.

    PubMed

    Miyagawa, Yoshitaka; Verlengia, Gianluca; Reinhart, Bonnie; Han, Fang; Uchida, Hiroaki; Zucchini, Silvia; Goins, William F; Simonato, Michele; Cohen, Justus B; Glorioso, Joseph C

    2017-09-15

    The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes.

  2. Genetic deletion of TNFRII gene enhances the Alzheimer-like pathology in an APP transgenic mouse model via reduction of phosphorylated IκBα.

    PubMed

    Jiang, Hong; He, Ping; Xie, Junxia; Staufenbiel, Matthias; Li, Rena; Shen, Yong

    2014-09-15

    Tumor necrosis factor receptor II (TNFRII) is one of the TNF receptor superfamily members and our recent pathological studies show that TNFRII is deficient in the brains of Alzheimer's disease (AD). However, the mechanisms of TNFRII in AD pathogenesis remain unclear. In the present study, by using the gene-targeting approach to delete TNFRII in AD transgenic mouse model, we found that, in the brain of APP23 mice with TNFRII deletion (APP23/TNFRII(-/-)), AD-like pathology, i.e. plaque formation and microglial activation, occurs as early as 6 months of age. To test whether the increased levels of Aβ plaques was due to elevated Aβ, we measured Aβ and found that Aβ levels indeed were significantly increased at this age. Because β-secretase, BACE1, is critical enzyme for Aβ production, we have examined BACE1 and found that BACE1 is increased in both protein levels and enzymatic activity as early as 6 months of age; Having shown that BACE1 promoter region contains NF-κB binding sites, we found that cytoplasmic NF-κB was elevated and SUMO1 binding to IκBα was decreased. To further verify these findings, we have overexpressed TNFRII and identified that overexpressing TNFRII can reverse the findings from APP23/TNFRII(-/-) mice. Altogether, our results demonstrate novel roles of TNFRII in the regulation of Aβ production, suggesting a potential therapeutic strategy for AD by up-regulating TNFRII levels and elevating phosphorylated IκBα by SUMOylation.

  3. Enhancing human-like collagen accumulation by deleting the major glucose transporter ptsG in recombinant Escherichia coli BL21.

    PubMed

    Luo, Yan'e; Zhang, Tao; Fan, Daidi; Mu, Tingzhen; Xue, Wenjiao; Hui, Junfeng; Ma, Xiaoxuan

    2014-01-01

    Collagen has been proven to be a valuable biomedical material for many medical applications. Human-like collagen (HLC) is a novel important biomedical material with diverse medical applications. In this work, recombinant Escherichia coli BL21 3.7 ∆ptsG was constructed, the characters of ptsG mutant strain were analyzed, and real-time quantitative polymerase chain reaction (PCR) was applied to investigate the effect of ptsG gene deletion on the transcriptional level of the phosphotransferase system (PTS) genes responsible for glucose transport. The HLC production and cell growth ability were 1.33- and 1.24-fold higher than those of its parent strain in the fermentation medium, respectively, and 1.16- and 1.17-fold in the modified minimal medium individually. The acetate accumulation decreased by 42%-56% compared to its parent strain in the fermentation medium, and 70%-87% in the modified minimal medium. The results of RT-qPCR showed that the transcriptional level of crr, ptsH, ptsI, and blgF in ptsG mutant all decreased dramatically, which inferred a decrease in the glucose uptake rate, but the transcriptional level of FruB and manX increased slightly, which demonstrated the activation of fructose- and mannose-specific transport pathways in the ptsG mutant. This study demonstrates that ptsG deletion is an effective strategy to reduce acetate accumulation and increase biomass and HLC production.

  4. Quantum deletion: Beyond the no-deletion principle

    SciTech Connect

    Adhikari, Satyabrata

    2005-11-15

    Suppose we are given two identical copies of an unknown quantum state and we wish to delete one copy from among the given two copies. The quantum no-deletion principle restricts us from perfectly deleting a copy but it does not prohibit us from deleting a copy approximately. Here we construct two types of a 'universal quantum deletion machine' which approximately deletes a copy such that the fidelity of deletion does not depend on the input state. The two types of universal quantum deletion machines are (1) a conventional deletion machine described by one unitary operator and (2) a modified deletion machine described by two unitary operators. Here it is shown that the modified deletion machine deletes a qubit with fidelity 3/4, which is the maximum limit for deleting an unknown quantum state. In addition to this we also show that the modified deletion machine retains the qubit in the first mode with average fidelity 0.77 (approx.) which is slightly greater than the fidelity of measurement for two given identical states, showing how precisely one can determine its state [S. Massar and S. Popescu, Phys. Rev. Lett. 74, 1259 (1995)]. We also show that the deletion machine itself is input state independent, i.e., the information is not hidden in the deleting machine, and hence we can delete the information completely from the deletion machine.

  5. Targeted chromosomal deletions and inversions in zebrafish.

    PubMed

    Gupta, Ankit; Hall, Victoria L; Kok, Fatma O; Shin, Masahiro; McNulty, Joseph C; Lawson, Nathan D; Wolfe, Scot A

    2013-06-01

    Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

  6. Targeted Deletion of the Antisilencer/Enhancer (ASE) Element from Intron 1 of the Myelin Proteolipid Protein Gene (Plp1) in Mouse Reveals that the Element Is Dispensable for Plp1 Expression in Brain during Development and Remyelination

    PubMed Central

    Pereira, Glauber B.; Meng, Fanxue; Kockara, Neriman T.; Yang, Baoli; Wight, Patricia A.

    2012-01-01

    Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. While removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is nonfunctional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. PMID:23157328

  7. Formulation of a mmaA4 Gene Deletion Mutant of Mycobacterium bovis BCG in Cationic Liposomes Significantly Enhances Protection against Tuberculosis

    PubMed Central

    Derrick, Steven C.; Dao, Dee; Yang, Amy; Kolibab, Kris; Jacobs, William R.; Morris, Sheldon L.

    2012-01-01

    A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) – D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe

  8. Deletion of fusion peptide or destabilization of fusion core of HIV gp41 enhances antigenicity and immunogenicity of 4E10 epitope

    SciTech Connect

    Li Jing; Chen Xi; Jiang Shibo Chen Yinghua

    2008-11-07

    The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformation as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.

  9. Free fatty acid production in the cyanobacterium Synechocystis sp. PCC 6803 is enhanced by deletion of the cyAbrB2 transcriptional regulator.

    PubMed

    Kawahara, Akihito; Sato, Yusuke; Saito, Yujiro; Kaneko, Yasuko; Takimura, Yasushi; Hagihara, Hiroshi; Hihara, Yukako

    2016-02-20

    The cyAbrB2 (Sll0822) transcriptional regulator in Synechocystis sp. PCC 6803 is involved in coordination of carbon and nitrogen metabolism and its deletion causes distinct phenotypes such as decreased expression levels of nitrogen-regulated genes and high accumulation of glycogen granules. From the viewpoint of metabolic engineering, the highly accumulated glycogen granules in the ΔcyabrB2 mutant could be a valuable source for the production of biofuels. Here, by disruption of the aas gene (slr1609) encoding acyl-acyl carrier protein synthetase and introduction of a gene encoding thioesterase from Umbellularia californica (UcTE), we conferred the ability of production and secretion of free fatty acids on the ΔcyabrB2 mutant. Notable features of the resulting ΔcyabrB2Δaas::UcTE strain compared with ΔcyabrB2 by RNA-seq analysis were decrease in expression levels of genes related to uptake and subsequent metabolism of nitrogen and carbon and increase in the expression level of sigE encoding a group 2 sigma factor. These changes in gene expression profile were not observed when the same genetic modification was introduced in the wild-type background. The ΔcyabrB2Δaas::UcTE strain showed two-folds higher free fatty acid productivity on a per OD730 basis compared with the Δaas::UcTE strain, without expense of the accumulated glycogen granules. This shows the potential of the ΔcyabrB2 mutant as the platform of biofuel production. The effective utilization of the accumulated glycogen must be the next task to be pursued. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Deletion of the Mucin-Like Molecule Muc1 Enhances Dendritic Cell Activation in Response to Toll-Like Receptor Ligands

    PubMed Central

    Williams, Marc A.; Bauer, Stephen; Lu, Wenju; Guo, Jia; Walter, Scott; Bushnell, Timothy P.; Lillehoj, Erik P.; Georas, Steve N.

    2010-01-01

    Dendritic cells (DC) are potent professional antigen-presenting cells that drive primary immune responses to infections or other agonists perceived as ‘dangerous’. Muc1 is the only cell surface mucin or MUC gene product that is expressed in DC. Unlike other members of this glycoprotein family, Muc1 possesses a unique cytosolic region capable of signal transduction and attenuating toll-like receptor (TLR) activation. The expression and function of Muc1 has been intensively investigated on epithelial and tumor cells, but relatively little is known about its function on DC. We hypothesized that Muc1 would influence in vitro generation and primary DC activation in response to the TLR4 and TLR5 ligands lipopolysaccharide and flagellin. Compared with Muc1+/+ DC, we found that Muc1−/− DC were constitutively activated, as determined by higher expression of co-stimulatory molecules (CD40, CD80 and CD86), greater secretion of immunoregulatory cytokines (TNF-α and VEGF), and better stimulation of allogeneic naïve CD4+ T cell proliferation. After activation by either LPS or flagellin and co-culture with allogeneic CD4+ T cells, Muc1−/− DC also induced greater secretion of TNF-α and IFN-γ compared to similarly activated Muc1+/+ DC. Taken together, our results indicate that deletion of Muc1 promotes a heightened functional response of DC in response to TLR4 and TLR5 signaling pathways, and suggests a previously under-appreciated role for Muc1 in regulating innate immune responses of DC. PMID:20375631

  11. Deletion of Kv4.2 gene eliminates dendritic A-type K+ current and enhances induction of long-term potentiation in hippocampal CA1 pyramidal neurons.

    PubMed

    Chen, Xixi; Yuan, Li-Lian; Zhao, Cuiping; Birnbaum, Shari G; Frick, Andreas; Jung, Wonil E; Schwarz, Thomas L; Sweatt, J David; Johnston, Daniel

    2006-11-22

    Dendritic, backpropagating action potentials (bAPs) facilitate the induction of Hebbian long-term potentiation (LTP). Although bAPs in distal dendrites of hippocampal CA1 pyramidal neurons are attenuated when propagating from the soma, their amplitude can be increased greatly via downregulation of dendritic A-type K+ currents. The channels that underlie these currents thus may represent a key regulatory component of the signaling pathways that lead to synaptic plasticity. We directly tested this hypothesis by using Kv4.2 knock-out mice. Deletion of the Kv4.2 gene and a loss of Kv4.2 protein resulted in a specific and near-complete elimination of A-type K+ currents from the apical dendrites of CA1 pyramidal neurons. The absence of dendritic Kv4.2-encoded A-type K+ currents led to an increase of bAP amplitude and an increase of concurrent Ca2+ influx. Furthermore, CA1 pyramidal neurons lacking dendritic A-type K+ currents from Kv4.2 knock-out mice exhibited a lower threshold than those of wild-type littermates for LTP induction with the use of a theta burst pairing protocol. LTP triggered with the use of a saturating protocol, on the other hand, remained indistinguishable between Kv4.2 knock-out and wild-type neurons. Our results support the hypothesis that dendritic A-type K+ channels, composed of Kv4.2 subunits, regulate action potential backpropagation and the induction of specific forms of synaptic plasticity.

  12. Adding and Deleting Images

    EPA Pesticide Factsheets

    Images are added via the Drupal WebCMS Editor. Once an image is uploaded onto a page, it is available via the Library and your files. You can edit the metadata, delete the image permanently, and/or replace images on the Files tab.

  13. Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance.

    PubMed

    Khair, Lyne; Chang, Ya-Ting; Subramanian, Lakxmi; Russell, Paul; Nakamura, Toru M

    2010-06-01

    While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

  14. Roles of the Checkpoint Sensor Clamp Rad9-Rad1-Hus1 (911)-Complex and the Clamp Loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe Telomere Maintenance

    PubMed Central

    Khair, Lyne; Chang, Ya-Ting; Subramanian, Lakxmi; Russell, Paul; Nakamura, Toru M.

    2011-01-01

    While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1Δ cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1Δ cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes. PMID:20505337

  15. The DNA binding domain of human DNA ligase I interacts with both nicked DNA and the DNA sliding clamps, PCNA and hRad9-hRad1-hHus1

    PubMed Central

    Song, Wei; Pascal, John M.; Ellenberger, Tom; Tomkinson, Alan E.

    2009-01-01

    The participation of the DNA ligase (hLigI) encoded by the human LIG1 gene in DNA replication and repair is mediated by an interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp. Interestingly, the catalytic fragment of hLigI encircles a DNA nick forming a ring that is similar in size and shape to the PCNA ring. Here we show that the DNA binding domain (DBD) within the hLigI catalytic fragment interacts with both PCNA and the heterotrimeric cell-cycle checkpoint clamp, hRad9-hRad1-hHus1 (9-1-1). The DBD preferentially binds to trimeric PCNA and the hRad1 subunit of 9-1-1. Unlike the majority of PCNA interacting proteins, the DBD does not interact with the interdomain connector loop region of PCNA but instead appears to interact with regions adjacent to the intersubunit interfaces within the PCNA trimer. Notably, the DBD not only binds specifically to DNA nicks but also mediates the formation of DNA protein complexes with PCNA. Based on these results, we suggest that the interface between the DBD and PCNA acts as a pivot facilitating the transition of the hLigI catalytic region fragment from an extended conformation to a ring structure when it engages a DNA nick. PMID:19523882

  16. Improvement of erythrose reductase activity, deletion of by-products and statistical media optimization for enhanced erythritol production from Yarrowia lipolytica mutant 49.

    PubMed

    Ghezelbash, Gholam Reza; Nahvi, Iraj; Emamzadeh, Rahman

    2014-08-01

    The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp(270) with Glu(270) in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.

  17. Deletion of EP4 on bone marrow-derived cells enhances inflammation and angiotensin II-induced abdominal aortic aneurysm formation.

    PubMed

    Tang, Eva H C; Shvartz, Eugenia; Shimizu, Koichi; Rocha, Viviane Z; Zheng, Chunyu; Fukuda, Daiju; Shi, Guo-Ping; Sukhova, Galina; Libby, Peter

    2011-02-01

    To examine whether a lack of prostaglandin E receptor 4 (EP4) on bone marrow-derived cells would increase local inflammation and enhance the formation of abdominal aortic aneurysm (AAA) in vivo. Prostaglandin E(2) (PGE(2)) through activation of EP4, can mute inflammation. Hypercholesterolemic low-density lipoprotein receptor knockout (LDLR(-/-)) mice transplanted with either EP4(+/+) (EP4(+/+)/LDLR(-/-)) or EP4(-/-) (EP4(-/-)/LDLR(-/-)) bone marrow received infusions of angiotensin II to induce AAA. Deficiency of EP4 on bone marrow-derived cells increased the incidence (50% of male EP4(+/+)/LDLR(-/-) mice versus 88.9% of male EP4(-/-)/LDLR(-/-) mice developed AAA; and 22% of female EP4(+/+)/LDLR(-/-) mice versus 83.3% of female EP4(-/-)/LDLR(-/-) mice developed AAA) and severity of AAA, increased monocyte chemoattractant protein-1 (2.72-fold in males and 1.64-fold in females), and enhanced infiltration of macrophages (3.8-fold in males and 2.44-fold in females) and T cells (1.88-fold in males and 1.66-fold in females) into AAA lesions. Lack of EP4 on bone marrow-derived cells augmented elastin fragmentation, increased apoptotic markers, and decreased smooth muscle cell accumulation within AAA lesions. Deficiency of EP4 on bone marrow-derived cells boosted inflammation and AAA formation induced by angiotensin II in hyperlipidemic mice. This study affirms the pathophysiologic importance of PGE(2) signaling through EP4 as an endogenous anti-inflammatory pathway involved in experimental aneurysm formation.

  18. Deletion (2)(q37)

    SciTech Connect

    Stratton, R.F.; Tolworthy, J.A.; Young, R.S.

    1994-06-01

    We report on a 5-month-old girl with widely spaced nipples, redundant nuchal skin, coarctation of the aorta, anal atresia with distal fistula, postnatal growth retardation, hypotonia, and sparse scalp hair. Initial clinical assessment suggested the diagnosis of Ullrich-Turner syndrome. Chromosome analysis showed a 46,XX,del(2)(q37) karyotype in peripheral lymphocytes. We compare her findings to those of other reported patients with terminal deletions of 2q. 8 refs., 2 figs., 1 tab.

  19. Heteroduplex Mapping of Heat-Resistant Deletion Mutants of Bacteriophage T5

    PubMed Central

    Scheible, Patricia P.; Rhoades, Marc

    1975-01-01

    The bacteriophage T5 is known to spontaneously generate deletion mutants (st mutants) exhibiting enhanced resistance to heat inactivation in citrate buffer. A series of such mutants has been isolated and the deletions visualized by electron microscopy of heteroduplex molecules. The deletions are found to cluster in one region of the chromosome. Images PMID:16789160

  20. C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

    PubMed Central

    Doroud, Delaram; Zahedifard, Farnaz; Vatanara, Alireza; Taslimi, Yasaman; Vahabpour, Rouholah; Torkashvand, Fatemeh; Vaziri, Behrooz; Rouholamini Najafabadi, Abdolhossein; Rafati, Sima

    2011-01-01

    Background We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. Methodology/Principal Findings Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b-CTE, pcDNA-cpa/b-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. Conclusions/Significance This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb-CTE cocktail vaccination against leishmaniasis so that the average

  1. Nature of frequent deletions in CEBPA.

    PubMed

    Fuchs, Ota; Kostecka, Arnost; Provaznikova, Dana; Krasna, Blazena; Brezinova, Jana; Filkukova, Jitka; Kotlin, Roman; Kouba, Michal; Kobylka, Petr; Neuwirtova, Radana; Jonasova, Anna; Caniga, Miroslav; Schwarz, Jiri; Markova, Jana; Maaloufova, Jacqueline; Sponerova, Dana; Novakova, Ludmila; Cermak, Jaroslav

    2009-01-01

    C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs to the family of leucine zipper transcription factors and is necessary for transcriptional control of granulocyte, adipocyte and hepatocyte differentiation, glucose metabolism and lung development. C/EBPalpha is encoded by an intronless gene. CEBPA mutations cause a myeloid differentiation block and were detected in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma and non-Hodgkin's lymphoma (NHL) patients. In this study we identified in 41 individuals from 824 screened individuals (290 AML patients, 382 MDS patients, 56 NHL patients and 96 healthy individuals) a single class of 23 deletions in CEBPA gene which involved a direct repeat of at least 2 bp. These mutations are characterised by the loss of one of two same repeats at the ends of deleted sequence. Three most frequent repeats included in these deletions in CEBPA gene are CGCGAG (493-498_865-870), GCCAAGCAGC (508-517_907-916) and GG (486-487_885-886), all according to GenBank accession no. NM_004364.2. A mechanism for deletion formation between two repetitive sequences can be recombination events in the repair process. Double-stranded cut in DNA can initiate these recombination events of adjacent DNA sequences.

  2. Homologous and homeologous intermolecular gene conversion are not differentially affected by mutations in the DNA damage or the mismatch repair genes RAD1, RAD50, RAD51, RAD52, RAD54, PMS1 and MSH2

    SciTech Connect

    Porter, G.; Westmoreland, J.; Priebe, S.

    1996-06-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad{sup +} vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. 67 refs., 5 figs., 4 tabs.

  3. Homologous and Homeologous Intermolecular Gene Conversion Are Not Differentially Affected by Mutations in the DNA Damage or the Mismatch Repair Genes Rad1, Rad50, Rad51, Rad52, Rad54, Pms1 and Msh2

    PubMed Central

    Porter, G.; Westmoreland, J.; Priebe, S.; Resnick, M. A.

    1996-01-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad(+) vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. PMID:8725224

  4. Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2

    PubMed Central

    2014-01-01

    Background In the adult central nervous system, axonal regeneration is abortive. Regulators of microtubule dynamics have emerged as attractive targets to promote axonal growth following injury as microtubule organization is pivotal for growth cone formation. In this study, we used conditioned neurons with high regenerative capacity to further dissect cytoskeletal mechanisms that might be involved in the gain of intrinsic axon growth capacity. Results Following a phospho-site broad signaling pathway screen, we found that in conditioned neurons with high regenerative capacity, decreased glycogen synthase kinase 3β (GSK3β) activity and increased microtubule growth speed in the growth cone were present. To investigate the importance of GSK3β regulation during axonal regeneration in vivo, we used three genetic mouse models with high, intermediate or no GSK3β activity in neurons. Following spinal cord injury, reduced GSK3β levels or complete neuronal deletion of GSK3β led to increased growth cone microtubule growth speed and promoted axon regeneration. While several microtubule-interacting proteins are GSK3β substrates, phospho-mimetic collapsin response mediator protein 2 (T/D-CRMP-2) was sufficient to decrease microtubule growth speed and neurite outgrowth of conditioned neurons and of GSK3β-depleted neurons, prevailing over the effect of decreased levels of phosphorylated microtubule-associated protein 1B (MAP1B) and through a mechanism unrelated to decreased levels of phosphorylated cytoplasmic linker associated protein 2 (CLASP2). In addition, phospho-resistant T/A-CRMP-2 counteracted the inhibitory myelin effect on neurite growth, further supporting the GSK3β-CRMP-2 relevance during axon regeneration. Conclusions Our work shows that increased microtubule growth speed in the growth cone is present in conditions of increased axonal growth, and is achieved following inactivation of the GSK3β-CRMP-2 pathway, enhancing axon regeneration through the glial scar

  5. Deletion of FoxO1 leads to shortening of QRS by increasing Na(+) channel activity through enhanced expression of both cardiac NaV1.5 and β3 subunit.

    PubMed

    Cai, Benzhi; Wang, Ning; Mao, Weike; You, Tao; Lu, Yan; Li, Xiang; Ye, Bo; Li, Faqian; Xu, Haodong

    2014-09-01

    Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na(+) channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na(+) channel activity in mouse ventricular cardiomyocytes and assess the cardiac electrophysiological phenotype of mice with cardiac FoxO1 deletion. Tamoxifen-induced and cardiac-specific FoxO1 deletion was confirmed by polymerase chain reaction (PCR). Cardiac FoxO1 deletion failed to result in either cardiac functional changes or hypertrophy as assessed by echocardiography and individual ventricular cell capacitances, respectively. Western blotting showed that FoxO1 was significantly decreased while NaV1.5 protein level was significantly increased in mouse hearts with FoxO1 deletion. Reverse transcription-PCR (RT-PCR) revealed that FoxO1 deletion led to an increase in NaV1.5 and Na(+) channel subunit β3 mRNA, but not β1, 2, and 4, or connexin 43. Whole patch-clamp recordings demonstrated that cardiac Na(+) currents were significantly augmented by FoxO1 deletion without affecting the steady-state activation and inactivation, leading to accelerated depolarization of action potentials in mouse ventricular cardiomyocytes. Electrocardiogram recordings showed that the QRS complex was significantly shortened and the P wave amplitude was significantly increased in conscious and unrestrained mice with cardiac FoxO1 deletion. NaV1.5 expression was decreased in the peri-infarct (border-zone) of mice with myocardial infarction and FoxO1 accumulated in the cardiomyocyte nuclei of chronic ischemic human hearts. Our findings indicate that FoxO1 plays an important role in the regulation of NaV1.5 and β3 subunit expressions as well as Na

  6. Genetic Counseling for the 22q11.2 Deletion

    ERIC Educational Resources Information Center

    McDonald-McGinn, Donna M.; Zackai, Elaine H.

    2008-01-01

    Because of advances in palliative medical care, children with the 22q11.2 deletion syndrome are surviving into adulthood. An increase in reproductive fitness will likely follow necessitating enhanced access to genetic counseling for these patients and their families. Primary care physicians/obstetric practitioners are in a unique position to…

  7. Genetic Counseling for the 22q11.2 Deletion

    ERIC Educational Resources Information Center

    McDonald-McGinn, Donna M.; Zackai, Elaine H.

    2008-01-01

    Because of advances in palliative medical care, children with the 22q11.2 deletion syndrome are surviving into adulthood. An increase in reproductive fitness will likely follow necessitating enhanced access to genetic counseling for these patients and their families. Primary care physicians/obstetric practitioners are in a unique position to…

  8. Spontaneous deletion formation within the beta-galactosidase gene of Lactobacillus bulgaricus.

    PubMed Central

    Mollet, B; Delley, M

    1990-01-01

    To investigate the genetic stability of the dairy organism Lactobacillus bulgaricus, we have analyzed 107 spontaneous mutations of the beta-galactosidase gene of this organism. Ten of these mutations were DNA rearrangements giving rise to different deletions, located predominantly within a small hot spot area. The DNA sequences of the different deletion junctions have been determined. The analysis showed that the deletions can be divided into two classes, depending on the presence of short direct-repeat sequences at the deletion endpoints and on the length of the deleted sequences. Possible mechanisms of these deletion formations and the involvement of inverted-repeat sequences that may enhance slipped DNA mispairing are discussed. Images PMID:2120187

  9. Deletion of ultraconserved elements yields viable mice

    SciTech Connect

    Ahituv, Nadav; Zhu, Yiwen; Visel, Axel; Holt, Amy; Afzal, Veena; Pennacchio, Len A.; Rubin, Edward M.

    2007-07-15

    Ultraconserved elements have been suggested to retainextended perfect sequence identity between the human, mouse, and ratgenomes due to essential functional properties. To investigate thenecessities of these elements in vivo, we removed four non-codingultraconserved elements (ranging in length from 222 to 731 base pairs)from the mouse genome. To maximize the likelihood of observing aphenotype, we chose to delete elements that function as enhancers in amouse transgenic assay and that are near genes that exhibit markedphenotypes both when completely inactivated in the mouse as well as whentheir expression is altered due to other genomic modifications.Remarkably, all four resulting lines of mice lacking these ultraconservedelements were viable and fertile, and failed to reveal any criticalabnormalities when assayed for a variety of phenotypes including growth,longevity, pathology and metabolism. In addition more targeted screens,informed by the abnormalities observed in mice where genes in proximityto the investigated elements had been altered, also failed to revealnotable abnormalities. These results, while not inclusive of all thepossible phenotypic impact of the deleted sequences, indicate thatextreme sequence constraint does not necessarily reflect crucialfunctions required for viability.

  10. Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion

    PubMed Central

    Polidori, Taisia; Palumbo, Emilio; Guigo, Roderic

    2017-01-01

    CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales. PMID:28253259

  11. Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion.

    PubMed

    Pulido-Quetglas, Carlos; Aparicio-Prat, Estel; Arnan, Carme; Polidori, Taisia; Hermoso, Toni; Palumbo, Emilio; Ponomarenko, Julia; Guigo, Roderic; Johnson, Rory

    2017-03-01

    CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.

  12. A constitutional 5q23 deletion.

    PubMed Central

    Rivera, H; Simi, P; Rossi, S; Pardelli, L; Di Paolo, M C

    1990-01-01

    A 14 month old girl was found to have a deletion of the whole of band 5q23. By comparing 19 other cases monosomic for a part of the 5q13-q31 segment, the constitutional 5q interstitial deletions fall into two groups: adult patients with Gardner-like symptoms and mental retardation associated with deletion 5q21-q22, and patients (mostly children) with unspecific signs and symptoms and different deletions. Images PMID:2325108

  13. Deletion 14q and pericentric inversion 14.

    PubMed Central

    Nielsen, J; Homma, A; Rasmussen, K; Ried, E; Sorensen, K; Saldana-Garcia, P

    1978-01-01

    A woman with deletion 14q as well as inversion 14 is presented, and physical signs are compared with those of patients with deletion long arm 13. No previous case of deletion long arm 14 has been published. Images PMID:671492

  14. Interstitial deletions are not the main mechanism leading to 18q deletions.

    PubMed Central

    Strathdee, G.; Harrison, W.; Riethman, H. C.; Goodart, S. A.; Overhauser, J.

    1994-01-01

    Most patients who present with the 18q- syndrome have an apparent terminal deletion of the long arm of chromosome 18. For precise phenotypic mapping of this syndrome, it is important to determine whether the deletions are terminal deletions or interstitial deletions. A human telomeric YAC clone has been identified that hybridizes specifically to the telomeric end of 18q. This clone was characterized and used to analyze seven patients with 18q deletions. By FISH and Southern blotting analysis, all patients were found to lack this chromosomal region on their deleted chromosome, demonstrating that the patients do not have cryptic interstitial deletions. Images Figure 2 Figure 1 Figure 3 PMID:8198131

  15. Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

    PubMed Central

    Rodríguez-Rubio, Lorena; Martínez, Beatriz; Rodríguez, Ana; Donovan, David M.

    2012-01-01

    Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections. PMID:22267667

  16. Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium.

    PubMed

    Moding, Everett J; Lee, Chang-Lung; Castle, Katherine D; Oh, Patrick; Mao, Lan; Zha, Shan; Min, Hooney D; Ma, Yan; Das, Shiva; Kirsch, David G

    2014-08-01

    Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

  17. Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein

    PubMed Central

    Jackson, Eleisha L.; Spielman, Stephanie J.

    2017-01-01

    Proteins evolve through two primary mechanisms: substitution, where mutations alter a protein’s amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single—amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein’s three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions. PMID:28369116

  18. Enhanced cellular content and lactate fraction of the poly(lactate-co-3-hydroxybutyrate) polyester produced in recombinant Escherichia coli by the deletion of σ factor RpoN.

    PubMed

    Kadoya, Ryosuke; Kodama, Yu; Matsumoto, Ken'ichiro; Taguchi, Seiichi

    2015-04-01

    A new approach at the transcriptional level was applied to lactate-based polyester production. Four σ factor disruptants, ΔrpoN, ΔrpoS, ΔfliA and ΔfecI, of Escherichia coli were used as hosts for poly(lactate-co-3-hydroxybutyrate) production from glucose. Among them, ΔrpoN caused dual positive effects of polymer production, enhanced cellular content and lactate fraction.

  19. Variable Deletion Strategies in Canonical Correlation Analysis.

    ERIC Educational Resources Information Center

    Tanguma, Jesus

    This paper presents three variable deletion strategies in canonical correlation analysis. All three strategies are illustrated by examples. The first strategy uses the canonical communality (h2) coefficients of the three functions to decide which variable to delete. The second function also uses the canonical communality coefficients, but only…

  20. Passenger Deletions Generate Therapeutic Vulnerabilities in Cancer

    PubMed Central

    Muller, Florian L.; Colla, Simona; Aquilanti, Elisa; Manzo, Veronica; Genovese, Giannicola; Lee, Jaclyn; Eisenson, Dan; Narurkar, Rujuta; Deng, Pingna; Nezi, Luigi; Lee, Michelle; Hu, Baoli; Hu, Jian; Sahin, Ergun; Ong, Derrick; Fletcher-Sananikone, Eliot; Ho, Dennis; Kwong, Lawrence; Brennan, Cameron; Wang, Y. Alan; Chin, Lynda; DePinho, Ronald A.

    2013-01-01

    Inactivation of tumor suppressor genes via homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighboring genes. We hypothesized that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities in the case where the collaterally deleted gene is a member of a functionally redundant family of genes exercising an essential function. The glycolytic gene Enolase 1 (ENO1) in the 1p36 locus is deleted in Glioblastoma (GBM), which is tolerated by expression of ENO2. We demonstrate that shRNA-mediated extinction of ENO2 selectively inhibits growth, survival, and tumorigenic potential of ENO1-deleted GBM cells and that the enolase inhibitor phosphonoacetohydroxamate (PhAH) is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger deleted genes encoding functionally-redundant essential activities and provide an effective treatment strategy for cancers harboring such genomic events. PMID:22895339

  1. A novel 3q29 deletion associated with autism, intellectual disability, psychiatric disorders, and obesity.

    PubMed

    Biamino, Elisa; Di Gregorio, Eleonora; Belligni, Elga Fabia; Keller, Roberto; Riberi, Evelise; Gandione, Marina; Calcia, Alessandro; Mancini, Cecilia; Giorgio, Elisa; Cavalieri, Simona; Pappi, Patrizia; Talarico, Flavia; Fea, Antonio M; De Rubeis, Silvia; Cirillo Silengo, Margherita; Ferrero, Giovanni Battista; Brusco, Alfredo

    2016-03-01

    Copy number variation (CNV) has been associated with a variety of neuropsychiatric disorders, including intellectual disability/developmental delay (ID/DD), autism spectrum disorder (ASD), and schizophrenia (SCZ). Often, individuals carrying the same pathogenic CNV display high clinical variability. By array-CGH analysis, we identified a novel familial 3q29 deletion (1.36 Mb), centromeric to the 3q29 deletion region, which manifests with variable expressivity. The deletion was identified in a 3-year-old girl diagnosed with ID/DD and autism and segregated in six family members, all affected by severe psychiatric disorders including schizophrenia, major depression, anxiety disorder, and personality disorder. All individuals carrying the deletion were overweight or obese, and anomalies compatible with optic atrophy were observed in three out of four cases examined. Amongst the 10 genes encompassed by the deletion, the haploinsufficiency of Optic Atrophy 1 (OPA1), associated with autosomal dominant optic atrophy, is likely responsible for the ophthalmological anomalies. We hypothesize that the haploinsufficiency of ATPase type 13A4 (ATP13A4) and/or Hairy/Enhancer of Split Drosophila homolog 1 (HES1) contribute to the neuropsychiatric phenotype, while HES1 deletion might underlie the overweight/obesity. In conclusion, we propose a novel contiguous gene syndrome due to a proximal 3q29 deletion variably associated with autism, ID/DD, psychiatric traits and overweight/obesity. © 2015 Wiley Periodicals, Inc.

  2. Interstitial deletions of chromosome 6q: genotype-phenotype correlation utilizing array CGH.

    PubMed

    Klein, O D; Cotter, P D; Moore, M W; Zanko, A; Gilats, M; Epstein, C J; Conte, F; Rauen, K A

    2007-03-01

    Interstitial deletions of the long arm of chromosome 6 are relatively rare, with fewer than 100 cases reported. Phenotypic variation is in large part due to differences in size and location of the segmental aneuploidy. We report three new patients with interstitial deletions of chromosome 6q defined at the molecular level by array comparative genomic hybridization (array CGH). In two of three cases, the molecular breakpoints differed from those indicated by conventional karyotyping, demonstrating the enhanced resolution of array CGH. Two patients had minimal deletions of 6 and 8.8 Mb involving 6q16.2-->q21, and the third patient had a deletion of 11.3 Mb spanning 6q15-->q21. All three had developmental delay, craniofacial dysmorphology, and functional eye disorders, suggesting that genes affecting brain and craniofacial development are located in 6q16.2-->q21, the deleted region common to all three patients. Furthermore, gene(s) for discordant phenotypic features, such as central diabetes insipidus, may reside at 6q15, the monosomic region unique to patient 3. All three cases described here showed loss of paternal alleles within the deleted segment, providing further evidence of the predominantly paternal origin for 6q deletions and rearrangements.

  3. Microarray-based ultra-high resolution discovery of genomic deletion mutations

    PubMed Central

    2014-01-01

    Background Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. Results Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. Conclusions Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence. PMID:24655320

  4. 1p36 deletion syndrome: an update

    PubMed Central

    Jordan, Valerie K; Zaveri, Hitisha P; Scott, Daryl A

    2015-01-01

    Deletions of chromosome 1p36 affect approximately 1 in 5,000 newborns and are the most common terminal deletions in humans. Medical problems commonly caused by terminal deletions of 1p36 include developmental delay, intellectual disability, seizures, vision problems, hearing loss, short stature, distinctive facial features, brain anomalies, orofacial clefting, congenital heart defects, cardiomyopathy, and renal anomalies. Although 1p36 deletion syndrome is considered clinically recognizable, there is significant phenotypic variation among affected individuals. This variation is due, at least in part, to the genetic heterogeneity seen in 1p36 deletions which include terminal and interstitial deletions of varying lengths located throughout the 30 Mb of DNA that comprise chromosome 1p36. Array-based copy number variant analysis can easily identify genomic regions of 1p36 that are deleted in an affected individual. However, predicting the phenotype of an individual based solely on the location and extent of their 1p36 deletion remains a challenge since most of the genes that contribute to 1p36-related phenotypes have yet to be identified. In addition, haploinsufficiency of more than one gene may contribute to some phenotypes. In this article, we review recent successes in the effort to map and identify the genes and genomic regions that contribute to specific 1p36-related phenotypes. In particular, we highlight evidence implicating MMP23B, GABRD, SKI, PRDM16, KCNAB2, RERE, UBE4B, CASZ1, PDPN, SPEN, ECE1, HSPG2, and LUZP1 in various 1p36 deletion phenotypes. PMID:26345236

  5. 1p36 deletion syndrome: an update.

    PubMed

    Jordan, Valerie K; Zaveri, Hitisha P; Scott, Daryl A

    2015-01-01

    Deletions of chromosome 1p36 affect approximately 1 in 5,000 newborns and are the most common terminal deletions in humans. Medical problems commonly caused by terminal deletions of 1p36 include developmental delay, intellectual disability, seizures, vision problems, hearing loss, short stature, distinctive facial features, brain anomalies, orofacial clefting, congenital heart defects, cardiomyopathy, and renal anomalies. Although 1p36 deletion syndrome is considered clinically recognizable, there is significant phenotypic variation among affected individuals. This variation is due, at least in part, to the genetic heterogeneity seen in 1p36 deletions which include terminal and interstitial deletions of varying lengths located throughout the 30 Mb of DNA that comprise chromosome 1p36. Array-based copy number variant analysis can easily identify genomic regions of 1p36 that are deleted in an affected individual. However, predicting the phenotype of an individual based solely on the location and extent of their 1p36 deletion remains a challenge since most of the genes that contribute to 1p36-related phenotypes have yet to be identified. In addition, haploinsufficiency of more than one gene may contribute to some phenotypes. In this article, we review recent successes in the effort to map and identify the genes and genomic regions that contribute to specific 1p36-related phenotypes. In particular, we highlight evidence implicating MMP23B, GABRD, SKI, PRDM16, KCNAB2, RERE, UBE4B, CASZ1, PDPN, SPEN, ECE1, HSPG2, and LUZP1 in various 1p36 deletion phenotypes.

  6. Mitochondrial DNA deletions sensitize cells to apoptosis at low heteroplasmy levels

    SciTech Connect

    Schoeler, S.; Szibor, R.; Gellerich, F.N.; Wartmann, T.; Mawrin, C.; Dietzmann, K.; Kirches, E. . E-mail: elmar.kirches@medizin.uni-magdeburg.de

    2005-06-24

    A heterogeneous group of multisystem disorders affecting various tissues and often including neuromuscular symptoms is caused by mutations of the mitochondrial genome, which codes 13 polypeptides of oxidative phosphorylation (OXPHOS) complexes and 22 tRNA genes needed for their translation. Since the link between OXPHOS dysfunction and clinical phenotype remains enigmatic in many diseases, a possible role of enhanced apoptosis is discussed besides bioenergetic crisis of affected cells. We analyzed the proapoptotic impact of the mitochondrial 5 kb common deletion (CD), affecting five tRNA genes, in transmitochondrial cybrid cell lines and found a slightly enhanced sensitivity to exogenous oxidative stress (H{sub 2}O{sub 2}) and a pronounced sensitization against death receptor stimulation (TRAIL) at a rather low CD heteroplasmy level of 22%. Mitochondrial deletions confer enhanced susceptibility against proapoptotic signals to proliferating cells, which might explain the elimination of deletions from hematopoietic stem cells.

  7. 78 FR 21916 - Procurement List; Proposed Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-12

    ... Commissaryagency (DECA), Fort Lee, VA Deletions The following products and service are proposed for deletion from... Activity: Defense Commissaryagency (DECA), Fort Lee, VA Barry S. Lineback, Director, Business...

  8. Dynamics of deletion genotypes in an experimental insect virus population

    PubMed Central

    Simón, Oihane; Williams, Trevor; Caballero, Primitivo; López-Ferber, Miguel

    2005-01-01

    Defective viruses, that are deficient in certain essential genes, are maintained in the population by transcomplementation, exploiting the gene products of complete genotypes in co-infected cells. This process becomes prevalent only when cells are frequently infected by several virus particles, and only then will the fitness of defective viruses be subjected to frequency-dependent selection. Deletion variants that are not infectious per os are present in a multicapsid nucleopolyhedrovirus (SfMNPV, Baculoviridae) that infects the fall armyworm, Spodoptera frugiperda. These variants enhance the pathogenicity and, therefore, the likelihood of transmission of the virus when co-infecting cells with complete genotypes, resulting in occlusion bodies (OBs) that may contain both genotypes co-occluded. Mixtures of complete (B) and defective (C) variants in ratios of 90% B+10% C, 50% B+50% C and 10% B+90% C were used to inoculate by injection S. frugiperda larvae. Viral OBs extracted from diseased insects were subjected to four or five successive rounds of per os infection. Following successive passages, genotype frequencies in all three experimental populations converged to a single equilibrium frequency comprising ∼20% of deletion genotype C and ∼80% of complete genotype B. This mirrors the relative proportions of deletion (22%) and complete (78%) genotypes observed in the wild-type SfMNPV population. The pathogenicity of experimental populations at the final passage was not significantly different from that of the wild-type isolate. In contrast, OBs of all genotype mixtures were significantly more pathogenic than OBs of genotype B alone. A population genetics model, in which virus populations were assigned linear frequency-dependent transmissibility values, was in remarkably close agreement to empirical data. Clearly, non-infectious deletion variants can profoundly affect the likelihood of transmission and the genetic structure and stability of virus populations. PMID

  9. 19 CFR 142.49 - Deletion of C-4 Code.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... director may temporarily or permanently delete an entry filer's C-4 Code without providing the participant.... Entry filers may delete C-4 Codes from Line Release by notifying the port director in writing on a Deletion Data Loading Sheet. Such notification shall state the C-4 Code which is to be deleted, the port...

  10. 19 CFR 142.49 - Deletion of C-4 Code.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... director may temporarily or permanently delete an entry filer's C-4 Code without providing the participant.... Entry filers may delete C-4 Codes from Line Release by notifying the port director in writing on a Deletion Data Loading Sheet. Such notification shall state the C-4 Code which is to be deleted, the port...

  11. 46 CFR 67.171 - Deletion; requirement and procedure.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Deletion; requirement and procedure. 67.171 Section 67...; Requirement for Exchange, Replacement, Deletion, Cancellation § 67.171 Deletion; requirement and procedure. (a... provided in § 67.161, and the vessel is subject to deletion from the roll of actively documented vessels...

  12. 46 CFR 67.171 - Deletion; requirement and procedure.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Deletion; requirement and procedure. 67.171 Section 67...; Requirement for Exchange, Replacement, Deletion, Cancellation § 67.171 Deletion; requirement and procedure. (a... provided in § 67.161, and the vessel is subject to deletion from the roll of actively documented vessels...

  13. 46 CFR 67.171 - Deletion; requirement and procedure.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Deletion; requirement and procedure. 67.171 Section 67...; Requirement for Exchange, Replacement, Deletion, Cancellation § 67.171 Deletion; requirement and procedure. (a... provided in § 67.161, and the vessel is subject to deletion from the roll of actively documented vessels...

  14. 46 CFR 67.171 - Deletion; requirement and procedure.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Deletion; requirement and procedure. 67.171 Section 67...; Requirement for Exchange, Replacement, Deletion, Cancellation § 67.171 Deletion; requirement and procedure. (a... provided in § 67.161, and the vessel is subject to deletion from the roll of actively documented vessels...

  15. 46 CFR 67.171 - Deletion; requirement and procedure.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Deletion; requirement and procedure. 67.171 Section 67...; Requirement for Exchange, Replacement, Deletion, Cancellation § 67.171 Deletion; requirement and procedure. (a... provided in § 67.161, and the vessel is subject to deletion from the roll of actively documented vessels...

  16. Myeloid Bmal1 deletion increases monocyte recruitment and worsens atherosclerosis.

    PubMed

    Huo, Mingyu; Huang, Yuhong; Qu, Dan; Zhang, Hongsong; Wong, Wing Tak; Chawla, Ajay; Huang, Yu; Tian, Xiao Yu

    2017-03-01

    BMAL1, the nonredundant transcription factor in the core molecular clock, has been implicated in cardiometabolic diseases in mice and humans. BMAL1 controls the cyclic trafficking of Ly6c(hi) monocytes to sites of acute inflammation. Myeloid deficiency of Bmal1 also worsens chronic inflammation in diet-induced obesity. We studied whether myeloid Bmal1 deletion promotes atherosclerosis by enhancing monocyte recruitment to atherosclerotic lesions. By generating Bmal1(FloxP/FloxP);LysM(Cre) mice on the Apoe(-/-) background, we showed that Bmal1 deletion in myeloid cells increased the size of atherosclerotic lesions. Bmal1 deficiency in monocytes and macrophages resulted in an increased total number of lesional macrophages in general and Ly6c(hi) infiltrating monocyte-macrophages in particular, accompanied by skewed M2 to M1 macrophage phenotype. Ly6c(hi) and/or Ly6c(lo) monocyte subsets in blood, spleen, and bone marrow were not altered. Cell tracking and adoptive transfer of Ly6c(hi) monocytes showed Bmal1 deficiency induced more trafficking of Ly6c(hi) monocytes to atherosclerotic lesions, preferential differentiation of Ly6c(hi) monocytes into M1 macrophages, and increased macrophage content and lesion size in the carotid arteries. We demonstrated that Bmal1 deficiency in macrophages promotes atherosclerosis by enhancing recruitment of Ly6c(hi) monocytes to atherosclerotic lesions.-Huo, M., Huang, Y., Qu, D., Zhang, H., Wong, W. T., Chawla, A., Huang, Y., Tian, X. Y. Myeloid Bmal1 deletion increases monocyte recruitment and worsens atherosclerosis.

  17. Deletion of zmp1 improves Mycobacterium bovis BCG-mediated protection in a guinea pig model of tuberculosis.

    PubMed

    Sander, Peter; Clark, Simon; Petrera, Agnese; Vilaplana, Cristina; Meuli, Michael; Selchow, Petra; Zelmer, Andrea; Mohanan, Deepa; Andreu, Nuria; Rayner, Emma; Dal Molin, Michael; Bancroft, Gregory J; Johansen, Pål; Cardona, Pere-Joan; Williams, Ann; Böttger, Erik C

    2015-03-10

    Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials.

  18. Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs.

    USDA-ARS?s Scientific Manuscript database

    Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene’s function in the living organism. In this protocol we describe the OSCAR method of precise and rapid deletion plasmid construction. OSCAR relies on t...

  19. STAT6 Deletion Enhances Immunity to Mammary Carcinoma

    DTIC Science & Technology

    2005-06-01

    CD34-FITC, CD16/ CD32 -FITC, rat IgG2a-PE isotype control, and rat were diluted in HEPES buffer (0.01 M, pH 7.35) with 2% FCS (HyClone). IgG2a-FITC...from STAT6-dcfi- WIIIj Cj08 cient mice express more CD16/ CD32 and CD80, whereas MSCs IJCD3 - i]I1 3 from BALB/c mice express more CDI Ic, DEC205, and

  20. Enhance, delete, incept: Manipulating hippocampus-dependent memories☆

    PubMed Central

    Spiers, Hugo J.; Bendor, Daniel

    2014-01-01

    Here we provide a brief overview of recent research on memory manipulation. We focus primarily on memories for which the hippocampus is thought to be required due to its central importance in the study of memory. The repertoire of methods employed is expanding and includes optogenetics, transcranial stimulation, deep brain stimulation, cued reactivation during sleep and the use of pharmacological agents. In addition, the possible mechanisms underlying these memory changes have been investigated using techniques such as single unit recording and functional magnetic resonance imaging (fMRI). This article is part of a Special Issue entitled ‘Memory enhancement’. PMID:24397964

  1. Somatic mosaicism for a DMD gene deletion

    SciTech Connect

    Saito, Kayoko; Ikeya, Kiyoko; Kondo, Eri

    1995-03-13

    Mosaicism is a mixed state, with two cell populations of different genetic origins caused by a cell mutation occurring after fertilization. In the present case, DNA analysis of lymphocytes led to a DMD diagnosis before death. Postmortem immunocytochemical and DNA analysis showed somatic mosaicism. At age 18 years, blood lymphocyte DNA analysis showed a DMD gene deletion, upstream from exon 7 to the 5{prime} end containing both muscle and brain promoters. As the patient`s mother and elder sister had no deletions, he was considered to have a new mutation. Immunocytochemical studies of postmortem tissues showed that dystrophin was absent from the tongue, deltoid, intercostal, psoas and rectus femoris muscles, but there was a mix of dystrophin-positive and negative fibers in the rectus abdominis, cardiac, temporalis and sternocleidomastoid muscles. All diaphragm cells were dystrophin positive. Polymerase chain reaction (PCR) amplification from all tissues except the temporalis and sternocleidomastoid muscles, diaphragm and kidney, in which no deletion was found, showed the deletion from at least exon 6 to the 5{prime} end containing both muscle and brain promoters. In this case, a genomic deletion of the DMD gene contributed to the formation of tissues derived from both ectoderm and endoderm, and cells of mesodermal origin showed genotypic and phenotypic heterogeneity. Our results indicate a mutation of the present case may have occurred just before the period of germ layer formation. 34 refs., 7 figs.

  2. Chromosome 7q31.1 deletion in myeloid neoplasms.

    PubMed

    Tripputi, Pasquale; Bianchi, Paola; Fermo, Elisa; Bignotto, Monica; Zanella, Alberto

    2014-02-01

    We studied monosomy and deletions of chromosome 7 in 208 patients with myeloid disorders; we found 39 patients (19%) with monosomy or deletion of chromosome 7: 24 patients with chromosome 7 deletion and 15 with monosomy 7. In the 24 patients with chromosome 7 deletions, studied with copy-number variants, short-tandem repeats, microsatellites, single nucleotide polymorphisms, and deletion polymorphisms, the most common deleted region was 7q31.1 (20 patients). Deletion polymorphism studies performed in these 20 patients showed an interstitial deletion of at least 140 kilobase in 6 patients; the deletion spans between the genes forkhead box P2 and Myo D family inhibitor domain containing. Because both genes do not seem to be involved in leukogenesis, we suggest to look carefully into this deletion for the presence of tumor suppressor genes and microRNAs. © 2014.

  3. Repeats, longevity and the sources of mtDNA deletions: evidence from “deletional spectra”

    PubMed Central

    Guo, Xinhong; Popadin, Konstantin Yu.; Markuzon, Natalya; Orlov, Yuriy L.; Kraytsberg, Yevgenya; Krishnan, Kim. J.; Zsurka, Gabor; Turnbull, Douglas M.; Kunz, Wolfram S.; Khrapko, Konstantin

    2010-01-01

    Perfect direct repeats and, in particular, the prominent 13-bp repeat, are thought to cause mitochondrial DNA (mtDNA) deletions, which have been associated with the aging process. Accordingly, individuals lacking the 13-bp repeat are highly prevalent among centenarians and the number of repeats negatively correlates with mammalian longevity. However, detailed examination of the distribution of mtDNA deletions challenges the role of the 13-bp repeat and other perfect repeats in generating mtDNA deletions. Instead, deletions appear to depend on long and stable, albeit imperfect, duplexes between distant mtDNA segments. Furthermore, significant dissimilarities in breakpoint distributions suggest that multiple mechanisms are involved in creating mtDNA deletions. PMID:20591530

  4. Monosomy 1p36 deletion syndrome.

    PubMed

    Gajecka, Marzena; Mackay, Katherine L; Shaffer, Lisa G

    2007-11-15

    Monosomy 1p36 results from a heterozygous deletion of the most distal chromosomal band on the short arm of chromosome 1. Occurring in approximately 1 in 5,000 live births, monosomy 1p36 is the most common terminal deletion observed in humans. Monosomy 1p36 is associated with mental retardation, developmental delay, hearing impairment, seizures, growth impairment, hypotonia, and heart defects. The syndrome is also characterized by several distinct dysmorphic features, including large anterior fontanels, microcephaly, brachycephaly, deep-set eyes, flat nose and nasal bridge, and pointed chin. Several genes have been proposed as causative for individual features of the phenotype. In addition, based upon molecular characterization of subjects with monosomy 1p36, several mechanisms for the generation and stabilization of terminal deletions have been proposed.

  5. Method for introducing unidirectional nested deletions

    DOEpatents

    Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

    2001-01-01

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment in the context of a cloning vector which contains an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment. Also disclosed is a method for producing single-stranded DNA probes utilizing the same cloning vector. An optimal vector, PZIP is described. Methods for introducing unidirectional deletions into a terminal location of a cloned DNA sequence which is inserted into the vector of the present invention are also disclosed. These methods are useful for introducing deletions into either or both ends of a cloned DNA insert, for high throughput sequencing of any DNA of interest.

  6. Selective neuronal PTEN deletion: can we take the brakes off of growth without losing control?

    PubMed

    Gutilla, Erin A; Steward, Oswald

    2016-08-01

    The limited ability for injured adult axons to regenerate is a major cause for limited functional recovery after injury to the nervous system, motivating numerous efforts to uncover mechanisms capable of enhancing regeneration potential. One promising strategy involves deletion or knockdown of the phosphatase and tensin (PTEN) gene. Conditional genetic deletion of PTEN before, immediately following, or several months after spinal cord injury enables neurons of the corticospinal tract (CST) to regenerate their axons across the lesion, which is accompanied by enhanced recovery of skilled voluntary motor functions mediated by the CST. Although conditional genetic deletion or knockdown of PTEN in neurons enables axon regeneration, PTEN is a well-known tumor suppressor and mutations of the PTEN gene disrupt brain development leading to neurological abnormalities including macrocephaly, seizures, and early mortality. The long-term consequences of manipulating PTEN in the adult nervous system, as would be done for therapeutic intervention after injury, are only now being explored. Here, we summarize evidence indicating that long-term deletion of PTEN in mature neurons does not cause evident pathology; indeed, cortical neurons that have lived without PTEN for over 1 year appear robust and healthy. Studies to date provide only a first look at potential negative consequences of PTEN deletion or knockdown, but the absence of any detectable neuropathology supports guarded optimism that interventions to enable axon regeneration after injury are achievable.

  7. Selective neuronal PTEN deletion: can we take the brakes off of growth without losing control?

    PubMed Central

    Gutilla, Erin A.; Steward, Oswald

    2016-01-01

    The limited ability for injured adult axons to regenerate is a major cause for limited functional recovery after injury to the nervous system, motivating numerous efforts to uncover mechanisms capable of enhancing regeneration potential. One promising strategy involves deletion or knockdown of the phosphatase and tensin (PTEN) gene. Conditional genetic deletion of PTEN before, immediately following, or several months after spinal cord injury enables neurons of the corticospinal tract (CST) to regenerate their axons across the lesion, which is accompanied by enhanced recovery of skilled voluntary motor functions mediated by the CST. Although conditional genetic deletion or knockdown of PTEN in neurons enables axon regeneration, PTEN is a well-known tumor suppressor and mutations of the PTEN gene disrupt brain development leading to neurological abnormalities including macrocephaly, seizures, and early mortality. The long-term consequences of manipulating PTEN in the adult nervous system, as would be done for therapeutic intervention after injury, are only now being explored. Here, we summarize evidence indicating that long-term deletion of PTEN in mature neurons does not cause evident pathology; indeed, cortical neurons that have lived without PTEN for over 1 year appear robust and healthy. Studies to date provide only a first look at potential negative consequences of PTEN deletion or knockdown, but the absence of any detectable neuropathology supports guarded optimism that interventions to enable axon regeneration after injury are achievable. PMID:27651754

  8. Deletion 22q13.3 syndrome.

    PubMed

    Phelan, Mary C

    2008-05-27

    The deletion 22q13.3 syndrome (deletion 22q13 syndrome or Phelan-McDermid syndrome) is a chromosome microdeletion syndrome characterized by neonatal hypotonia, global developmental delay, normal to accelerated growth, absent to severely delayed speech, and minor dysmorphic features. The deletion occurs with equal frequency in males and females and has been reported in mosaic and non-mosaic forms. Due to lack of clinical recognition and often insufficient laboratory testing, the syndrome is under-diagnosed and its true incidence remains unknown. Common physical traits include long eye lashes, large or unusual ears, relatively large hands, dysplastic toenails, full brow, dolicocephaly, full cheeks, bulbous nose, and pointed chin. Behavior is autistic-like with decreased perception of pain and habitual chewing or mouthing. The loss of 22q13.3 can result from simple deletion, translocation, ring chromosome formation and less common structural changes affecting the long arm of chromosome 22, specifically the region containing the SHANK3 gene. The diagnosis of deletion 22q13 syndrome should be considered in all cases of hypotonia of unknown etiology and in individuals with absent speech. Although the deletion can sometimes be detected by high resolution chromosome analysis, fluorescence in situ hybridization (FISH) or array comparative genomic hybridization (CGH) is recommended for confirmation. Differential diagnosis includes syndromes associated with hypotonia, developmental delay, speech delay and/or autistic-like affect (Prader-Willi, Angelman, Williams, Smith-Magenis, Fragile X, Sotos, FG, trichorhinophalangeal and velocardiofacial syndromes, autism spectrum disorders, cerebral palsy). Genetic counseling is recommended and parental laboratory studies should be considered to identify cryptic rearrangements and detect parental mosaicism. Prenatal diagnosis should be offered for future pregnancies in those families with inherited rearrangements. Individuals with

  9. Phenotypic variability in Angelman syndrome: comparison among different deletion classes and between deletion and UPD subjects.

    PubMed

    Varela, Monica Castro; Kok, Fernando; Otto, Paulo Alberto; Koiffmann, Celia Priszkulnik

    2004-12-01

    Angelman syndrome (AS) can result from either a 15q11-q13 deletion (del), paternal uniparental disomy (UPD), imprinting, or UBE3A mutations. Here, we describe the phenotypic and behavioral variability detected in 49 patients with different classes of deletions and nine patients with UPD. Diagnosis was made by methylation pattern analysis of exon 1 of the SNRPN-SNURF gene and by microsatellite profiling of loci within and outside the 15q11-q13 region. There were no major phenotypic differences between the two main classes (BP1-BP3; BP2-BP3) of AS deletion patients, except for the absence of vocalization, more prevalent in patients with BP1-BP3 deletions, and for the age of sitting without support, which was lower in patients with BP2-BP3 deletions. Our data suggest that gene deletions (NIPA1, NIPA2, CYF1P1, GCP5) mapped to the region between breakpoints BP1 and BP2 may be involved in the severity of speech impairment, since all BP1-BP3 deletion patients showed complete absence of vocalization, while 38.1% of the BP2-BP3 deletion patients were able to pronounce syllabic sounds, with doubtful meaning. Compared to UPD patients, deletion patients presented a higher incidence of swallowing disorders (73.9% del x 22.2% UPD) and hypotonia (73.3% del x 28.57% UPD). In addition, children with UPD showed better physical growth, fewer or no seizures, a lower incidence of microcephaly, less ataxia and higher cognitive skills. As a consequence of their milder or less typical phenotype, AS may remain undiagnosed, leading to an overall underdiagnosis of the disease.

  10. Deletion 5q35.3

    SciTech Connect

    Stratton, R.F.; Tedrowe, N.A.; Tolworthy, J.A.; Patterson, R.M.; Ryan, S.G.; Young, R.S.

    1994-06-01

    The authors report on a 15-month-old boy with a de novo deletion of the terminal band of 5q, macrocephaly, mild retrognathia, anteverted nares with low flat nasal bridge, telecanthus, minor earlobe anomalies, bellshaped chest, diastasis recti, short fingers, and mild developmental delay.

  11. Interstitial deletion (6)q13q15

    SciTech Connect

    Gershoni-Baruch, R.; Mandel, H.; Bar El, H.; Bar-Nizan, N.; Borochowitz, Z.; Dar, Hanna

    1996-04-24

    We report on a 2-year-old child with psychomotor retardation, facial and urogenital anomalies. His chromosome constitution was 46,XY,del(6)(q13q15). This case further contributes to the karyotype-phenotype correlation of proximal deletion 6q syndromes. 18 refs., 3 figs., 1 tab.

  12. 22q11 deletion syndrome: current perspective

    PubMed Central

    Hacıhamdioğlu, Bülent; Hacıhamdioğlu, Duygu; Delil, Kenan

    2015-01-01

    Chromosome 22q11 is characterized by the presence of chromosome-specific low-copy repeats or segmental duplications. This region of the chromosome is very unstable and susceptible to mutations. The misalignment of low-copy repeats during nonallelic homologous recombination leads to the deletion of the 22q11.2 region, which results in 22q11 deletion syndrome (22q11DS). The 22q11.2 deletion is associated with a wide variety of phenotypes. The term 22q11DS is an umbrella term that is used to encompass all 22q11.2 deletion-associated phenotypes. The haploinsufficiency of genes located at 22q11.2 affects the early morphogenesis of the pharyngeal arches, heart, skeleton, and brain. TBX1 is the most important gene for 22q11DS. This syndrome can ultimately affect many organs or systems; therefore, it has a very wide phenotypic spectrum. An increasing amount of information is available related to the pathogenesis, clinical phenotypes, and management of this syndrome in recent years. This review summarizes the current clinical and genetic status related to 22q11DS. PMID:26056486

  13. Chromosomal deletions in the myelodysplastic syndrome.

    PubMed

    Mufti, G J

    1992-01-01

    Karyotypic abnormalities in primary myelodysplastic syndrome (P-MDS) are less frequent than in secondary myelodysplasia. A review of the literature involving over 3000 reported cases, shows the incidence of karyotypically abnormal clones at presentation in nearly 48% of cases. Approximately 50% of the abnormalities comprise of deletions of chromosomes 5, 7, 11, 12, 13 and 20. Localisation of a number of haemopoietic growth factors and their receptors to the deleted segments of the chromosomes, has invoked considerable interest in the molecular pathology of the interstitial deletions and their consequent role in the multistep pathogenesis of MDS. Present evidence suggests chromosome abnormalities are a later event in the multistep painogenesis, and it is suggested their occurrence may be restricted to a restricted myeloid progenitor cell, although the initial event(s) occur at the common lymphoid-myeloid progenitor. Much has been gleaned from the dominant modes of leukaemogenesis, such as the occurrence of missense mutations at specific positions of RAS and FMS mutations. It is suggested that a similar enquiry into the mechanisms of chromosomal deletions in P-MDS is required in order to delineate the role of these abnormalities in the clonal evolution of this group of diseases.

  14. Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions

    PubMed Central

    Ombrello, Michael J.; Remmers, Elaine F.; Sun, Guangping; Freeman, Alexandra F.; Datta, Shrimati; Torabi-Parizi, Parizad; Subramanian, Naeha; Bunney, Tom D.; Baxendale, Rhona W.; Martins, Marta S.; Romberg, Neil; Komarow, Hirsh; Aksentijevich, Ivona; Kim, Hun Sik; Ho, Jason; Cruse, Glenn; Jung, Mi-Yeon; Gilfillan, Alasdair M.; Metcalfe, Dean D.; Nelson, Celeste; O'Brien, Michelle; Wisch, Laura; Stone, Kelly; Douek, Daniel C.; Gandhi, Chhavi; Wanderer, Alan A.; Lee, Hane; Nelson, Stanley F.; Shianna, Kevin V.; Cirulli, Elizabeth T.; Goldstein, David B.; Long, Eric O.; Moir, Susan; Meffre, Eric; Holland, Steven M.; Kastner, Daniel L.; Katan, Matilda; Hoffman, Hal M.; Milner, Joshua D.

    2012-01-01

    Background Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. Methods We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. Results Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ2 (PLCγ2), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. Conclusions Genomic deletions in PLCG2 cause gain of PLCγ2 function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded

  15. Ku80-deletion suppresses spontaneous tumors and induces a p53-mediated DNA damage response

    PubMed Central

    Holcomb, Valerie B.; Rodier, Francis; Choi, Yong Jun; Busuttil, Rita A.; Vogel, Hannes; Vijg, Jan; Campisi, Judith; Hasty, Paul

    2014-01-01

    Ku80 facilitates DNA repair and therefore should suppress cancer. However, ku80−/− mice exhibit reduced cancer, although they age prematurely and have a shortened life span. We tested the hypothesis that Ku80 deletion suppresses cancer by enhancing cellular tumor suppressive responses to inefficiently repaired DNA damage. In support of this hypothesis, Ku80 deletion ameliorated tumor burden in APCMIN mice, and increased a p53-mediated DNA damage response, DNA lesions, and chromosomal rearrangements. Thus, contrary to its assumed role as a caretaker tumor suppressor, Ku80 facilitates tumor growth most likely by dampening baseline cellular DNA damage responses. PMID:19010925

  16. Deletional Protein Engineering Based on Stable Fold

    PubMed Central

    Sokalingam, Sriram; Yun, Hyungdon; Lee, Sun-Gu

    2012-01-01

    Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability. PMID:23240034

  17. 5p14 deletion associated with microcephaly and seizures

    PubMed Central

    Johnson, E.; Marinescu, R; Punnett, H.; Tenenholz, B.; Overhauser, J.

    2000-01-01

    We report on a father and son who have an interstitial deletion of 5p14. The father is clinically and mentally normal while the son has significant clinical involvement including microcephaly, seizures, and global developmental delay. The extent of the 5p14 deletion was determined using fluorescence in situ hybridisation (FISH). The deletion in this present family is smaller than a deletion previously described in a multigenerational family that lacks any clinical phenotype. This report shows that a 5p14 deletion does not always lead to a normal phenotype.


Keywords: interstitial deletion; chromosome 5; fluorescence in situ hybridisation; cri du chat syndrome PMID:10662813

  18. Improving freeze-tolerance of baker's yeast through seamless gene deletion of NTH1 and PUT1.

    PubMed

    Dong, Jian; Chen, Didi; Wang, Guanglu; Zhang, Cuiying; Du, Liping; Liu, Shanshan; Zhao, Yu; Xiao, Dongguang

    2016-06-01

    Baker's yeast strains with freeze-tolerance are highly desirable to maintain high leavening ability after freezing. Enhanced intracellular concentration of trehalose and proline in yeast is linked with freeze-tolerance. In this study, we constructed baker's yeast with enhanced freeze-tolerance by simultaneous deletion of the neutral trehalase-encoded gene NTH1 and the proline oxidase-encoded gene PUT1. We first used the two-step integration-based seamless gene deletion method to separately delete NTH1 and PUT1 in haploid yeast. Subsequently, through two rounds of hybridization and sporulation-based allelic exchange and colony PCR-mediated tetrad analysis, we obtained strains with restored URA3 and deletion of NTH1 and/or PUT1. The resulting strain showed higher cell survival and dough-leavening ability after freezing compared to the wild-type strain due to enhanced accumulation of trehalose and/or proline. Moreover, mutant with simultaneous deletion of NTH1 and PUT1 exhibits the highest relative dough-leavening ability after freezing compared to mutants with single-gene deletion perhaps due to elevated levels of both trehalose and proline. These results verified that it is applicable to construct frozen dough baker's yeast using the method proposed in this paper.

  19. Conditional deletion of ELL2 induces murine prostate intraepithelial neoplasia.

    PubMed

    Pascal, Laura E; Masoodi, Khalid Z; Liu, June; Qiu, Xiaonan; Song, Qiong; Wang, Yujuan; Zang, Yachen; Yang, Tiejun; Wang, Yao; Rigatti, Lora H; Chandran, Uma; Colli, Leandro M; Vencio, Ricardo Z N; Lu, Yi; Zhang, Jian; Wang, Zhou

    2017-11-01

    Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivoEll2-knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2-knockout mice. Microarray analysis of prostates from Ell2-knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer. © 2017 Society for Endocrinology.

  20. Recurring exon deletions in the haptoglobin (HP) gene associate with lower blood cholesterol levels

    PubMed Central

    Boettger, Linda M.; Salem, Rany M.; Handsaker, Robert E.; Peloso, Gina; Kathiresan, Sekar; Hirschhorn, Joel; McCarroll, Steven A.

    2016-01-01

    Two exons of the human haptoglobin (HP) gene exhibit copy number variation that affects HP multimerization and underlies one of the first protein polymorphisms identified in humans. The evolutionary origins and medical significance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from the recurring reversion of an ancient hominin-specific duplication of these exons. Though this polymorphism has been largely invisible to genome-wide genetic studies to date, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We show that these deletions, and a SNP that affects HP expression, are the likely drivers of the strong but complex association of cholesterol levels to SNPs near HP. Recurring exonic deletions in the haptoglobin gene likely enhance human health by lowering cholesterol levels in the blood. PMID:26901066

  1. Deletions and candidate genes in Williams syndrome

    SciTech Connect

    Perez Jurado, L.A.; Peoples, R.; Francke, U.

    1994-09-01

    Hemizygosity at the elastin locus (ELN) on chromosome 7q11.23 has recently been reported in several familial and sporadic cases of the developmental disorder, Williams syndrome (WS). Because the deletion is greater than the span of the ELN gene, a contiguous gene deletion syndrome has been suggested as the probable molecular basis for this condition. Thus far, neither the size of the deletion(s), nor other genes within it are known. We have analyzed samples from 27 sporadic WS patients by genotyping two multiallelic ELN intragenic polymorphisms, detectable by PCR amplification, and by Southern blotting for ELN gene dosage. Twenty four patients were hemizygous at the ELN locus while 3 showed no deletion or detectable rearrangement. Genotype studies on parental DNA were informative in 12 of the deletions. All 12 were due to de novo events, 8 in the maternal and 4 in the paternal chromosome. In an attempt to identify genes involved in WS we are also using a candidate gene approach. Delayed clearance of an exogenous calcium load with normal or slightly increased calcitonin levels in serum has been documented in WS patients suggesting a defective calcitonin action or calcium sensing function. The calcitonin receptor (CTR) gene is, therefore, a good candidate since CTR has a dual role as a hormonal receptor for calcitonin and an extracellular calcium sensor. We have mapped the CTR gene to chromosome 7q21.1 by PCR-SSCA of somatic cell hybrids and FISH analysis. Using two color FISH with probes for ELN and CTR, both loci are located on 7q at a distance of {approximately}10 Mb, CTR being telomeric. Our CTR probe does not detect any genomic abnormality by FISH or Southern blot in the patients` samples analyzed. We have identified a diallelic polymorphism in the CTR cDNA and are currently testing the hypothesis of an impaired CTR expression as responsible for some of the clinical features of WS by analysing the CTR transcripts by RT-PCR.

  2. Genetics Home Reference: distal 18q deletion syndrome

    MedlinePlus

    ... B, O'Donnell L, Gelfond J, Lancaster J, Fox PT, Hale DE. Consequences of chromsome18q deletions. Am ... Cody JD, Andrews T, Hardies LJ, Hale DE, Fox PT. Myelination in children with partial deletions of ...

  3. 49 CFR 7.6 - Deletion of identifying detail.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... necessary to prevent a clearly unwarranted invasion of personal privacy, identifying details will be deleted... withheld by another Federal statute, such information shall be deleted from any record covered by this...

  4. 49 CFR 7.6 - Deletion of identifying detail.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... necessary to prevent a clearly unwarranted invasion of personal privacy, identifying details will be deleted... withheld by another Federal statute, such information shall be deleted from any record covered by this...

  5. 49 CFR 7.6 - Deletion of identifying detail.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... necessary to prevent a clearly unwarranted invasion of personal privacy, identifying details will be deleted... withheld by another Federal statute, such information shall be deleted from any record covered by this...

  6. In-Frame Deletions Allow Functional Characterization of Complex Cellulose Degradation Phenotypes in Cellvibrio japonicus.

    PubMed

    Nelson, Cassandra E; Gardner, Jeffrey G

    2015-09-01

    The depolymerization of the recalcitrant polysaccharides found in lignocellulose has become an area of intense interest due to the role of this process in global carbon cycling, human gut microbiome nutritional contributions, and bioenergy production. However, underdeveloped genetic tools have hampered study of bacterial lignocellulose degradation, especially outside model organisms. In this report, we describe an in-frame deletion strategy for the Gram-negative lignocellulose-degrading bacterium Cellvibrio japonicus. This method leverages optimized growth conditions for conjugation and sacB counterselection for the generation of markerless in-frame deletions. This method produces mutants in as few as 8 days and allows for the ability to make multiple gene deletions per strain. It is also possible to remove large sections of the genome, as shown in this report with the deletion of the nine-gene (9.4-kb) gsp operon in C. japonicus. We applied this system to study the complex phenotypes of cellulose degradation in C. japonicus. Our data indicated that a Δcel5B Δcel6A double mutant is crippled for cellulose utilization, more so than by either single mutation alone. Additionally, we deleted individual genes in the two-gene cbp2ED operon and showed that both genes contribute to cellulose degradation in C. japonicus. Overall, these described techniques substantially enhance the utility of C. japonicus as a model system to study lignocellulose degradation.

  7. Effects of G-gene Deletion and Replacement on Rabies Virus Vector Gene Expression

    PubMed Central

    Sato, Sho; Ohara, Shinya; Tsutsui, Ken-Ichiro; Iijima, Toshio

    2015-01-01

    The glycoprotein-gene (G gene) -deleted rabies virus (RV) vector is a powerful tool to examine the function and structure of neural circuits. We previously reported that the deletion of the G gene enhances the transgene expression level of the RV vector. However, the mechanism of this enhancement remains to be clarified. We presume that there are two possible factors for this enhancement. The first factor is the glycoprotein of RV, which shows cytotoxicity; thus, may cause a dysfunction in the translation process of infected cells. The second possible factor is the enhanced expression of the L gene, which encodes viral RNA polymerase. In the RV, it is known that the gene expression level is altered depending on the position of the gene. Since G-gene deletion displaces the L gene in the genome, the expression of the L gene and viral transcription may be enhanced. In this study, we compared the transgene expression level and viral transcription of three recombinant RV vectors. The effect of glycoprotein was examined by comparing the viral gene expression of G-gene-intact RV and G-gene-replaced RV. Despite the fact that the L-gene transcription level of these two RV vectors was similar, the G-gene-replaced RV vector showed higher viral transcription and transgene expression level than the G-gene-intact RV vector. To examine the effect of the position of the L gene, we compared the viral gene expression of the G-gene-deleted RV and G-gene-replaced RV. The G-gene-deleted RV vector showed higher L-gene transcription, viral transcription, and transgene expression level than the G-gene-replaced RV vector. These results indicate that G-gene deletion enhances the transgene expression level through at least two factors, the absence of glycoprotein and enhancement of L-gene expression. These findings enable investigators to design a useful viral vector that shows a controlled desirable transgene expression level in applications. PMID:26023771

  8. Depth perception from dynamic occlusion in motion parallax: Roles of expansion-compression versus accretion-deletion

    PubMed Central

    Yoonessi, Ahmad; Baker, Curtis L.

    2013-01-01

    Motion parallax, or differential retinal image motion from observer movement, provides important information for depth perception. We previously measured the contribution of shear motion parallax to depth, which is only composed of relative motion information. Here, we examine the roles of relative motion and accretion-deletion information in dynamic occlusion motion parallax. Observers performed two-alternative forced choice depth-ordering tasks in response to low spatial frequency patterns of horizontal random dot motion that were synchronized to the observer's head movements. We examined conditions that isolated or combined expansion-compression and accretion-deletion across a range of simulated relative depths. At small depths, expansion-compression provided reliable depth perception while accretion-deletion had a minor contribution: When the two were in conflict, the perceived depth was dominated by expansion-compression. At larger depths in the cue-conflict experiment, accretion-deletion determined the depth-ordering performance. Accretion-deletion in isolation did not yield any percept of depth even though, in theory, it provided sufficient information for depth ordering. Thus, accretion-deletion can substantially enhance depth perception at larger depths but only in the presence of relative motion. The results indicate that expansion-compression contributes to depth from motion parallax across a broad range of depths whereas accretion-deletion contributes primarily at larger depths. PMID:24130259

  9. Depth perception from dynamic occlusion in motion parallax: roles of expansion-compression versus accretion-deletion.

    PubMed

    Yoonessi, Ahmad; Baker, Curtis L

    2013-10-15

    Motion parallax, or differential retinal image motion from observer movement, provides important information for depth perception. We previously measured the contribution of shear motion parallax to depth, which is only composed of relative motion information. Here, we examine the roles of relative motion and accretion-deletion information in dynamic occlusion motion parallax. Observers performed two-alternative forced choice depth-ordering tasks in response to low spatial frequency patterns of horizontal random dot motion that were synchronized to the observer's head movements. We examined conditions that isolated or combined expansion-compression and accretion-deletion across a range of simulated relative depths. At small depths, expansion-compression provided reliable depth perception while accretion-deletion had a minor contribution: When the two were in conflict, the perceived depth was dominated by expansion-compression. At larger depths in the cue-conflict experiment, accretion-deletion determined the depth-ordering performance. Accretion-deletion in isolation did not yield any percept of depth even though, in theory, it provided sufficient information for depth ordering. Thus, accretion-deletion can substantially enhance depth perception at larger depths but only in the presence of relative motion. The results indicate that expansion-compression contributes to depth from motion parallax across a broad range of depths whereas accretion-deletion contributes primarily at larger depths.

  10. Duplication/deletion of chromosome 8p

    SciTech Connect

    Priest, J.H.

    1995-09-11

    The article by Guo et al. provides evidence for deletion of D8S596 loci (assigned to 8p23) in at least some patients with inverted duplications of 8p. Cytogenetic break points forming the inverted duplication are remarkably similar among most of their patients and those reported previously, suggesting a common mechanism for this interesting rearrangement. Why should similar breaks occur in 8p and why is a FISH signal absent in the distal short arm when the ONCOR digoxigenin-labeled probe for loci D8S596 is used? Other studies also indicate that duplication for the region 8p12-p22 is associated with a deletion distal to the duplication itself. 4 refs.

  11. 19 CFR 142.49 - Deletion of C-4 Code.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 19 Customs Duties 2 2013-04-01 2013-04-01 false Deletion of C-4 Code. 142.49 Section 142.49... TREASURY (CONTINUED) ENTRY PROCESS Line Release § 142.49 Deletion of C-4 Code. (a) By Customs. A port director may temporarily or permanently delete an entry filer's C-4 Code without providing the participant...

  12. Creating, Searching, and Deleting KD Trees Using C++

    DTIC Science & Technology

    2014-09-01

    Creating, Searching, and Deleting KD Trees Using C++ by Robert J Yager ARL-TN-0629 September 2014...Deleting KD Trees Using C++ by Robert J Yager Weapons and Materials Research Directorate, ARL...Searching, and Deleting KD Trees Using C++ 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Robert J Yager

  13. Investigation of NRXN1 deletions: clinical and molecular characterization.

    PubMed

    Dabell, Mindy Preston; Rosenfeld, Jill A; Bader, Patricia; Escobar, Luis F; El-Khechen, Dima; Vallee, Stephanie E; Dinulos, Mary Beth Palko; Curry, Cynthia; Fisher, Jamie; Tervo, Raymond; Hannibal, Mark C; Siefkas, Kiana; Wyatt, Philip R; Hughes, Lauren; Smith, Rosemarie; Ellingwood, Sara; Lacassie, Yves; Stroud, Tracy; Farrell, Sandra A; Sanchez-Lara, Pedro A; Randolph, Linda M; Niyazov, Dmitriy; Stevens, Cathy A; Schoonveld, Cheri; Skidmore, David; MacKay, Sara; Miles, Judith H; Moodley, Manikum; Huillet, Adam; Neill, Nicholas J; Ellison, Jay W; Ballif, Blake C; Shaffer, Lisa G

    2013-04-01

    Deletions at 2p16.3 involving exons of NRXN1 are associated with susceptibility for autism and schizophrenia, and similar deletions have been identified in individuals with developmental delay and dysmorphic features. We have identified 34 probands with exonic NRXN1 deletions following referral for clinical microarray-based comparative genomic hybridization. To more firmly establish the full phenotypic spectrum associated with exonic NRXN1 deletions, we report the clinical features of 27 individuals with NRXN1 deletions, who represent 23 of these 34 families. The frequency of exonic NRXN1 deletions among our postnatally diagnosed patients (0.11%) is significantly higher than the frequency among reported controls (0.02%; P = 6.08 × 10(-7) ), supporting a role for these deletions in the development of abnormal phenotypes. Generally, most individuals with NRXN1 exonic deletions have developmental delay (particularly speech), abnormal behaviors, and mild dysmorphic features. In our cohort, autism spectrum disorders were diagnosed in 43% (10/23), and 16% (4/25) had epilepsy. The presence of NRXN1 deletions in normal parents and siblings suggests reduced penetrance and/or variable expressivity, which may be influenced by genetic, environmental, and/or stochastic factors. The pathogenicity of these deletions may also be affected by the location of the deletion within the gene. Counseling should appropriately represent this spectrum of possibilities when discussing recurrence risks or expectations for a child found to have a deletion in NRXN1. Copyright © 2013 Wiley Periodicals, Inc.

  14. Characterizing Deletion Transformations across Dialects using a Sophisticated Tying Mechanism

    DTIC Science & Technology

    2011-03-30

    suggest nrle candidates for further linguistic studies. Potential appli- cations include forensic phonetics, accent training, and dialect recognition...03-2011 Technical Paper MAR 2011 - APR 2011 Characterizing Deletion Transformations across Dialects using a Sophisticated Tying Mechanism FA8720-05...modeling deletion transformations between dialects . We empirically show that the proposed tying mechanism reduces deletion errors by 33% when compared to a

  15. Characterization of five partial deletions of the factor VIII gene

    SciTech Connect

    Youssoufian, H.; Antonarakis, S.E.; Aronis, S.; Tsiftis, G.; Phillips, D.G.; Kazazian, H.H. Jr.

    1987-06-01

    Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, the authors have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes.

  16. Chromosome 11q13 deletion syndrome

    PubMed Central

    Kim, Yu-Seon; Kim, Gun-Ha; Byeon, Jung Hye; Eun, So-Hee

    2016-01-01

    Chromosome 11q13 deletion syndrome has been previously reported as either otodental syndrome or oculo-oto-dental syndrome. The otodental syndrome is characterized by dental abnormalities and high-frequency sensorineural hearing loss, and by ocular coloboma in some cases. The underlying genetic defect causing otodental syndrome is a hemizygous microdeletion involving the FGF3 gene on chromosome 11q13.3. Recently, a new form of severe deafness, microtia (small ear) and small teeth, without the appearance of eye abnormalities, was also reported. In this report, we describe a 1-year-old girl presenting with ptosis of the left upper eyelid, right auricular deformity, high-arched palate, delayed dentition, simian line on the right hand, microcephaly, and developmental delay. In this patient, we identified a deletion in the chromosome 11q13.2-q13.3 (2.75 Mb) region by using an array-comparative genomic hybridization analysis. The deletion in chromosome 11q13 results in a syndrome characterized by variable clinical manifestations. Some of these manifestations involve craniofacial dysmorphology and require a functional workup for hearing, ophthalmic examinations, and long-term dental care. PMID:28018436

  17. Deletion Diagnostics for Alternating Logistic Regressions

    PubMed Central

    Preisser, John S.; By, Kunthel; Perin, Jamie; Qaqish, Bahjat F.

    2013-01-01

    Deletion diagnostics are introduced for the regression analysis of clustered binary outcomes estimated with alternating logistic regressions, an implementation of generalized estimating equations (GEE) that estimates regression coefficients in a marginal mean model and in a model for the intracluster association given by the log odds ratio. The diagnostics are developed within an estimating equations framework that recasts the estimating functions for association parameters based upon conditional residuals into equivalent functions based upon marginal residuals. Extensions of earlier work on GEE diagnostics follow directly, including computational formulae for one-step deletion diagnostics that measure the influence of a cluster of observations on the estimated regression parameters and on the overall marginal mean or association model fit. The diagnostic formulae are evaluated with simulations studies and with an application concerning an assessment of factors associated with health maintenance visits in primary care medical practices. The application and the simulations demonstrate that the proposed cluster-deletion diagnostics for alternating logistic regressions are good approximations of their exact fully iterated counterparts. PMID:22777960

  18. Immune function in patients with chromosome deletions.

    PubMed Central

    Nurmi, T; Uhari, M; Linna, S L; Silvennoinen-Kassinen, S; Koskela, M; Kiuttu, J; Tiilikainen, A

    1982-01-01

    Non-specific, cell-mediated and humoral immunity were evaluated in six patients with different autosomal deletions, and in two patients with X-chromosome deletions. Six had an increased number of bacterial, viral, and mycotic infections. Mild disturbances were found in the immunological functions of almost every patient. Granulocyte phagocytosis and killing of bacteria were normal in all patients. The chemotactic response was increased in two, and normal in the others. The responses to phytohaemagglutinin and pokeweed mitogen were normal in all patients and the response to concanavalin A was decreased in one patient. The lymphocyte response to purified protein derivative was decreased in the patients as a group when compared to the controls (P less than 0 . 005), but normal to oidiomycin. The number of acid-alpha-naphthyl acetate esterase positive cells was low in four patients. One had a high titre of antinuclear and antithyroid antibodies. One had a low concentration of serum IgA, C3 and C4. One had a high concentration of IgM. Two had elevated levels of C3 and C4. Our results show that several different chromosomal deletions are associated with immunological abnormality. PMID:6979446

  19. U-insertion/deletion Edited Sequence Database.

    PubMed

    Simpson, L; Wang, S H; Thiemann, O H; Alfonzo, J D; Maslov, D A; Avila, H A

    1998-01-01

    Uridine insertion/deletion RNA editing is a post-transcriptional RNA modification occurring in the mitochondria of kinetoplastid protozoa. The U-insertion/deletion Edited Sequence Database is a compilation of mitochondrial genes and edited mRNAs from five kinetoplastid species. It contains separate files with the DNA, mRNA (both unedited and edited) and predicted protein sequences, as well as alignments of the Leishmania tarentolae and Trypanosoma brucei protein sequences from edited and unedited genes. The sequence files are in GCG format. A 'map' sequence file showing the location of U-deletions, U-insertions and the translated amino acid sequences is also provided for each gene. Genomic maps for each species are also provided with clickable genes, including maxicircle-encoded gRNAs. Sets of aligned nuclear rRNA sequences from kinetoplastid protozoa are also provided, which were used for phylogenetic reconstructions in an analysis of the origin of RNA editing. The database is available through the World Wide Web as an HTML document at the URLhttp://www.lifesci.ucla.edu/RNA/trypanosome/ database.html

  20. Molecular refinement of the 1p36 deletion syndrome reveals size diversity and a preponderance of maternally derived deletions.

    PubMed

    Wu, Y Q; Heilstedt, H A; Bedell, J A; May, K M; Starkey, D E; McPherson, J D; Shapira, S K; Shaffer, L G

    1999-02-01

    The deletion of chromosome 1p36 is a newly recognized, relatively common contiguous gene deletion syndrome with a variable phenotype. The clinical features have recently been delineated and molecular analysis indicates that the prevalence of certain phenotypic features appears to correlate with deletion size. Phenotype/genotype comparisons have allowed the assignment of certain clinical features to specific deletion intervals, significantly narrowing the regions within which to search for candidate genes. We have extensively characterized the deletion regions in 30 cases using microsatellite markers and fluorescence in situ hybridization analyses. The map order of 28 microsatellite markers spanning the deletion region was obtained by a combination of genotypic analysis and physical mapping. The deletion region was divided into six intervals and breakpoints were found to cluster in mainly two regions. Molecular analysis of the deletions showed that two patients had complex re-arrangements; these cases shared their distal and proximal breakpoints in the two common breakpoint regions. Of the de novo deletions ( n = 28) in whichparental samples were available and the analysis was informative ( n = 27), there were significantly morematernally derived deletions ( n = 21) than paternally derived deletions ( n = 6) (chi1(2) = 8.35, P < 0.0001). Phenotype/genotype correlations and refinements of critical regions in our naturally occurring deletion panel have delineated specific areas in which to focus the search for the causative genes for the features of this syndrome.

  1. Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans.

    PubMed

    Peters, Björn; Junker, Anja; Brauer, Katharina; Mühlthaler, Bernadette; Kostner, David; Mientus, Markus; Liebl, Wolfgang; Ehrenreich, Armin

    2013-03-01

    Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on D-mannitol, D-fructose or in the presence of L-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.

  2. Deletion of nudB causes increased susceptibility to antifolates in Escherichia coli and Salmonella enterica.

    PubMed

    Li, Kun; Li, Ting; Yang, Shan-Shan; Wang, Xu-De; Gao, Lei-Xin; Wang, Rui-Qi; Gu, Jing; Zhang, Xian-En; Deng, Jiao-Yu

    2017-02-21

    Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium, and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7, 8-dihydroneopterin and 7, 8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on the susceptibility to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects the susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in the increased susceptibility to both SMX and TMP in Salmonella enterica Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be

  3. PHLPP1 gene deletion protects the brain from ischemic injury

    PubMed Central

    Chen, Bo; Van Winkle, Jessica A; Lyden, Patrick D; Brown, Joan H; Purcell, Nicole H

    2013-01-01

    A recently discovered protein phosphatase PHLPP (PH domain Leucine-rich repeat Protein Phosphatase) has been shown to dephosphorylate Akt on its hydrophobic motif (Ser473) thereby decreasing Akt kinase activity. We generated PHLPP1 knockout (KO) mice and used them to explore the ability of enhanced in vivo Akt signaling to protect the brain against ischemic insult. Brains from KO mice subjected to middle cerebral artery occlusion (MCAO) for 2 hours showed significantly greater increases in Akt activity and less neurovascular damage after reperfusion than wild-type (WT) mice. Remarkably, infarct volume in the PHLPP1 KO was significantly reduced compared with WT (12.7±2.7% versus 22.9±3.1%) and this was prevented by Akt inhibition. Astrocytes from KO mice and neurons in which PHLPP1 was downregulated showed enhanced Akt activation and diminished cell death in response to oxygen-glucose deprivation. Thus, deletion of PHLPP1 can enhance Akt activation in neurons and astrocytes, and can significantly increase cell survival and diminish infarct size after MCAO. Inhibition of PHLPP could be a therapeutic approach to minimize damage after focal ischemia. PMID:23072745

  4. Phenotypic characterization of rare interstitial deletion of chromosome 4

    PubMed Central

    Ismail, Samira; Helmy, Nivine A.; Mahmoud, Wael M.; El-Ruby, Mona O.

    2012-01-01

    Interstitial deletion of the long arm of chromosome 4 is rare. Patients with interstitial deletion of the long arm of chromosome 4 differ from those with terminal deletions. Phenotypes may be variable, depending upon the specific length and location of the deleted portion. Here, we report on a boy exhibiting most of the congenital malformations encountered in terminal 4q syndrome. The conventional karyotyping and Fluorescence in-situ hybridization revealed a de novo interstitial del (4)(q31q32). The current report is a further document highlighting that deletion of segment q31 could be contributing to the expression of most of the phenotype of 4q deletion syndrome. Using array comparative genome hybridization methodology is recommended for investigating further cases with similar segmental interstitial deletions to support and delineate findings and to define genes implicated in the pathogenesis of the disorder. PMID:27625821

  5. Conditional Deletion of Ferritin H in Mice Reduces B and T Lymphocyte Populations

    PubMed Central

    Vanoaica, Liviu; Richman, Larry; Jaworski, Maike; Darshan, Deepak; Luther, Sanjiv A.; Kühn, Lukas C.

    2014-01-01

    The immune system and iron availability are intimately linked as appropriate iron supply is needed for cell proliferation, while excess iron, as observed in hemochromatosis, may reduce subsets of lymphocytes. We have tested the effects of a ferritin H gene deletion on lymphocytes. Mx-Cre mediated conditional deletion of ferritin H in bone marrow reduced the number of mature B cells and peripheral T cells in all lymphoid organs. FACS analysis showed an increase in the labile iron pool, enhanced reactive oxygen species formation and mitochondrial depolarization. The findings were confirmed by a B-cell specific deletion using Fthlox/lox; CD19-Cre mice. Mature B cells were strongly under-represented in bone marrow and spleen of the deleted mice, whereas pre-B and immature B cells were not affected. Bone marrow B cells showed increased proliferation as judged by the number of cells in S and G2/M phase as well as BrdU incorporation. Upon in vitro culture with B-cell activating factor of the tumor necrosis factor family (BAFF), ferritin H-deleted spleen B cells showed lower survival rates than wild type cells. This was partially reversed with iron-chelator deferiprone. The loss of T cells was also confirmed by a T cell-specific deletion in Fthlox/lox;CD4-Cre mice. Our data show that ferritin H is required for B and T cell survival by actively reducing the labile iron pool. They further suggest that natural B and T cell maturation is influenced by intracellular iron levels and possibly deregulated in iron excess or deprivation. PMID:24586648

  6. Submicroscopic deletions at 22q11.2: Variability of the clinical picture and delineation of a commonly deleted region

    SciTech Connect

    Lindsay, E.A.; Shaffer, L.G.; Greenberg, F.

    1995-03-27

    DiGeorge anomaly (DGA) and velo-cardio-facial syndrome (VCFS) are frequently associated with monosomy of chromosome region 22q11. Most patients have a submicroscopic deletion, recently estimated to be at least 1-2 Mb. It is not clear whether individuals who present with only some of the features of these conditions have the deletion, and if so, whether the size of the deletion varies from those with more classic phenotypes. We have used fluorescence in situ hybridization (FISH) to assess the deletion status of 85 individuals referred to us for molecular analysis, with a wide range of DGA-like or VCFS-like clinical features. The test probe used was the cosmid sc11.1, which detects two loci about 2 Mb apart in 22q11.2. Twenty-four patients carried the deletion. Of the deleted patients, most had classic DGA or VCFS phenotypes, but 6 deleted patients had mild phenotypes, including 2 with minor facial anomalies and velopharyngeal incompetence as the only presenting signs. Despite the great phenotypic variability among the deleted patients, none had a deletion smaller than the 2-Mb region defined by sc11.1. Smaller deletions were not detected in patients with particularly suggestive phenotypes who were not deleted for sc11.1, even when tested with two other probes from the DGA/VCFS region. 24 refs., 2 figs., 2 tabs.

  7. Targeted gene deletion in Zygosaccharomyces bailii.

    PubMed

    Mollapour, M; Piper, P

    2001-01-30

    Yeasts of the genus Zygosaccharomyces are notable agents of large-scale food spoilage. Despite the economic importance of these organisms, little is known about the stress adaptations whereby they adapt to many of the more severe conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservatives and ethanol, can be isolated by complementation of the corresponding mutant strains of Saccharomyces cerevisiae. It was also discovered that the acquisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was sufficient for the former yeast to acquire the ability to degrade two major food preservatives, benzoic acid and sorbic acid. Using DNA cassettes containing dominant selectable markers and methods originally developed for performing gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. bailii genome were sequentially deleted. The resulting Zbyme2/Zbyme2 homozygous deletant strain had lost any ability to utilize benzoate as sole carbon source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono-oxygenase function, is essential for Z. bailii to degrade food preservatives. This ability to catabolize weak acid preservatives is a significant factor contributing to the preservative resistance of Z. bailii under aerobic conditions. This study is the first to demonstrate that it is possible to delete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of further mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Accession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170).

  8. Writing and deleting single magnetic skyrmions.

    PubMed

    Romming, Niklas; Hanneken, Christian; Menzel, Matthias; Bickel, Jessica E; Wolter, Boris; von Bergmann, Kirsten; Kubetzka, André; Wiesendanger, Roland

    2013-08-09

    Topologically nontrivial spin textures have recently been investigated for spintronic applications. Here, we report on an ultrathin magnetic film in which individual skyrmions can be written and deleted in a controlled fashion with local spin-polarized currents from a scanning tunneling microscope. An external magnetic field is used to tune the energy landscape, and the temperature is adjusted to prevent thermally activated switching between topologically distinct states. Switching rate and direction can then be controlled by the parameters used for current injection. The creation and annihilation of individual magnetic skyrmions demonstrates the potential for topological charge in future information-storage concepts.

  9. CD36 deletion improves recovery from spinal cord injury

    PubMed Central

    Myers, Scott A.; Andres, Kariena R.; Hagg, Theo; Whittemore, Scott R.

    2014-01-01

    CD36 is a pleiotropic receptor involved in several pathophysiological conditions, including cerebral ischemia, neurovascular dysfunction and atherosclerosis, and recent reports implicate its involvement in the endoplasmic reticulum stress response (ERSR). We hypothesized that CD36 signaling contributes to the inflammation and microvascular dysfunction following spinal cord injury. Following contusive injury, CD36−/− mice demonstrated improved hindlimb functional recovery and greater white matter sparing than CD36+/+ mice. CD36−/− mice exhibited a reduced macrophage, but not neutrophil, infiltration into the injury epicenter. Fewer infiltrating macrophages were either apoptotic or positive for the ERSR marker, phospho-ATF4. CD36−/− mice also exhibited significant improvements in injury heterodomain vascularity and function. These microvessels accumulated less of the oxidized lipid product 4-hydroxy-trans-2-nonenal (4HNE) and exhibited a reduced ERSR, as detected by vascular phospho-ATF4, CHOP and CHAC-1 expression. In cultured primary endothelial cells, deletion of CD36 diminished 4HNE-induced phospho-ATF4 and CHOP expression. A reduction in phospho-eIF2α and subsequent increase in KDEL-positive, ER-localized proteins suggest that 4HNE-CD36 signaling facilitates the detection of misfolded proteins upstream of eIF2α phosphorylation, ultimately leading to CHOP-induced apoptosis. We conclude that CD36 deletion modestly, but significantly, improves functional recovery from spinal cord injury by enhancing vascular function and reducing macrophage infiltration. These phenotypes may, in part, stem from reduced ER stress-induced cell death within endothelial and macrophage cells following injury. PMID:24690303

  10. Presence of Large Deletions in Kindreds with Autism

    PubMed Central

    Yu, Chang-En; Dawson, Geraldine; Munson, Jeffrey; D’Souza, Ian; Osterling, Julie; Estes, Annette; Leutenegger, Anne-Louise; Flodman, Pamela; Smith, Moyra; Raskind, Wendy H.; Spence, M. Anne; McMahon, William; Wijsman, Ellen M.; Schellenberg, Gerard D.

    2002-01-01

    Autism is caused, in part, by inheritance of multiple interacting susceptibility alleles. To identify these inherited factors, linkage analysis of multiplex families is being performed on a sample of 105 families with two or more affected sibs. Segregation patterns of short tandem repeat polymorphic markers from four chromosomes revealed null alleles at four marker sites in 12 families that were the result of deletions ranging in size from 5 to >260 kb. In one family, a deletion at marker D7S630 was complex, with two segments deleted (37 kb and 18 kb) and two retained (2,836 bp and 38 bp). Three families had deletions at D7S517, with each family having a different deletion (96 kb, 183 kb, and >69 kb). Another three families had deletions at D8S264, again with each family having a different deletion, ranging in size from <5.9 kb to >260 kb. At a fourth marker, D8S272, a 192-kb deletion was found in five families. Unrelated subjects and additional families without autism were screened for deletions at these four sites. Families screened included 40 families from Centre d'Etude du Polymorphisme Humaine and 28 families affected with learning disabilities. Unrelated samples were 299 elderly control subjects, 121 younger control subjects, and 248 subjects with Alzheimer disease. The deletion allele at D8S272 was found in all populations screened. For the other three sites, no additional deletions were identified in any of the groups without autism. Thus, these deletions appear to be specific to autism kindreds and are potential autism-susceptibility alleles. An alternative hypothesis is that autism-susceptibility alleles elsewhere cause the deletions detected here, possibly by inducing errors during meiosis. PMID:12058345

  11. AZF deletions in infertile men from the Republic of Macedonia.

    PubMed

    Plaseski, Toso; Novevski, Predrag; Kocevska, Borka; Dimitrovski, Cedomir; Efremov, Georgi D; Plaseska-Karanfilska, Dijana

    2006-07-01

    Y chromosome deletions in the three azoospermia factor (AZF) regions constitute the most common genetic cause of spermatogenic failure. The aim of this study was to estimate the length and boundaries of the AZF deletions and to correlate the AZF deletions with the sperm concentrations, testicular histology, Y haplogroups and the ethnic origin of the men with deletions. PCR analysis of STS loci in the three AZF regions was used to characterize the deletions. Y haplogroup was predicted from a set of 17 Y short tandem repeats (STR) marker values. A total of nine men out of 218 infertile/subfertile men showed the presence of Y microdeletions. In eight patients the results were consistent with the presence of AZFc deletions, while in one patient a larger deletion involving both AZFb and AZFc regions was detected. In two patients, the deletion, initially diagnosed as AZFc, involved part of the distal part of the AZFb region and in one of them the deletion also extended into the region distal to the AZFc. The 3.5 Mb AZFc deletion, due to homologous recombination between b2 and b4 amplicons, was detected in six men (66.7% of all Y deletions), thus being the most common type of AZF deletion among infertile men from the Republic of Macedonia. Patients with the 3.5 Mb AZFc deletion had azoospermia or severe oligozoospermia and variable histological results [Sertoly cell only syndrome (SCOS), maturity arrest (MA) and hypospermatogenesis (HSG)]. They were of different ethnic origin (Macedonian, Albanian and Romany) and belonged to different Y haplogroups (I1b, J2, E3b and G).

  12. Method for introducing unidirectional nested deletions

    DOEpatents

    Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

    1999-07-27

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

  13. Method for introducing unidirectional nested deletions

    DOEpatents

    Dunn, J.J.; Quesada, M.A.; Randesi, M.

    1999-07-27

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector. The cloning vector has an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe. 1 fig.

  14. Arginase II Deletion Increases Corpora Cavernosa Relaxation in Diabetic Mice

    PubMed Central

    Toque, Haroldo; Tostes, Rita; Yao, Lin; Xu, Zhimin; Webb, Clinton R.; Caldwell, Ruth; Caldwell, Robert

    2010-01-01

    Introduction Diabetes-induced erectile dysfunction involves elevated arginase (Arg) activity and expression. Because nitric oxide (NO) synthase and Arg share and compete for their substrate L-arginine, NO production is likely linked to regulation of Arg. Arg is highly expressed and implicated in erectile dysfunction. Aim It was hypothesized that Arg-II isoform deletion enhances relaxation function of corpora cavernosal (CC) smooth muscle in a streptozotocin (STZ) diabetic model. Methods Eight weeks after STZ-induced diabetes, vascular functional studies, Arg activity assay, and protein expression levels of Arg and constitutive NOS (using western blots) were assessed in CC tissues from non-diabetic wild type (WT), diabetic (D) WT (WT+D), Arg-II knockout (KO) and Arg-II KO+D mice (N=8–10 per group). Main Outcome Measures Inhibition or lack of arginase results in facilitation of CC relaxation in diabetic CC. Results Strips of CC from Arg-II KO mice exhibited an enhanced maximum endothelium-dependent relaxation (from 70+3% to 84+4%) and increased nitrergic relaxation (by 55%, 71%, 42%, 42%, and 24% for 1, 2, 4, 8 and 16 Hz, respectively) compared to WT mice. WT+D mice showed a significant reduction of endothelium-dependent maximum relaxation (44+8%), but this impairment of relaxation was significantly prevented in Arg-II KO+D mice (69+4%). Sympathetic-mediated and alpha-adrenergic agent-induced contractile responses also were increased in CC strips from D compared to non-D controls. Contractile responses were significantly lower in Arg-II KO control and D versus the WT groups. WT+D mice increased Arg activity (1.5-fold) and Arg-II protein expression and decreased total and phospho-eNOS at Ser-1177, and nNOS levels. These alterations were not seen in Arg-II KO mice. Additionally, the Arg inhibitor BEC (50 μM) enhanced nitrergic and endothelium-dependent relaxation in CC of WT+D mice. Conclusion These studies show for the first time that Arg-II deletion improves CC

  15. Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment

    PubMed Central

    2012-01-01

    Background Hepatitis B virus (HBV), because of its error-prone viral polymerase, has a high mutation rate leading to widespread substitutions, deletions, and insertions in the HBV genome. Deletions may significantly change viral biological features complicating the progression of liver diseases. However, the clinical conditions correlating to the accumulation of deleted mutants remain unclear. In this study, we explored HBV deletion patterns and their association with disease status and antiviral treatment by performing whole genome sequencing on samples from 51 hepatitis B patients and by monitoring changes in deletion variants during treatment. Clone sequencing was used to analyze preS regions in another cohort of 52 patients. Results Among the core, preS, and basic core promoter (BCP) deletion hotspots, we identified preS to have the highest frequency and the most complex deletion pattern using whole genome sequencing. Further clone sequencing analysis on preS identified 70 deletions which were classified into 4 types, the most common being preS2. Also, in contrast to the core and BCP regions, most preS deletions were in-frame. Most deletions interrupted viral surface epitopes, and are possibly involved in evading immuno-surveillance. Among various clinical factors examined, logistic regression showed that antiviral medication affected the accumulation of deletion mutants (OR = 6.81, 95% CI = 1.296 ~ 35.817, P = 0.023). In chronic carriers of the virus, and individuals with chronic hepatitis, the deletion rate was significantly higher in the antiviral treatment group (Fisher exact test, P = 0.007). Particularly, preS2 deletions were associated with the usage of nucleos(t)ide analog therapy (Fisher exact test, P = 0.023). Dynamic increases in preS1 or preS2 deletions were also observed in quasispecies from samples taken from patients before and after three months of ADV therapy. In vitro experiments demonstrated that preS2 deletions alone

  16. Are there ethnic differences in deletions in the dystrophin gene?

    SciTech Connect

    Banerjee, M.; Verma, I.C.

    1997-01-20

    We studied 160 cases of Duchenne muscular dystrophy (DMD) drawn from all parts of India, using multiplex PCR of 27 exons. Of these, 103 (64.4%) showed intragenic deletions. Most (69.7%) of the deletions involved exons 45-51. The phenotype of cases with deletion of single exons did not differ significantly from those with deletion of multiple exons. The distribution of deletions in studies from different countries was variable, but this was accounted for either by the small number of cases studied, or by fewer exons analyzed. It is concluded that there is likely to be no ethnic difference with respect to deletions in the DMD gene. 38 refs., 2 figs., 3 tabs.

  17. Genomic deletions suggest a phylogeny for the Mycobacterium tuberculosis complex.

    PubMed

    Mostowy, Serge; Cousins, Debby; Brinkman, Jacqui; Aranaz, Alicia; Behr, Marcel A

    2002-07-01

    To better understand the evolution of the Mycobacterium tuberculosis complex, subspecies were tested for large sequence polymorphisms. Samples with greater numbers of deletions, without exception, were missing all the same regions that were deleted from samples with lesser numbers of deletions. Principal genetic groups based on single-nucleotide polymorphisms were restricted to one of the deletion-based groups, and isolates that shared genotypes based on molecular epidemiological markers were assigned almost exclusively to the same deletion type. The data provide compelling evidence that human tuberculosis did not originate from the present-day bovine form. Genomic deletions present themselves as an attractive modality to study the evolution of the M. tuberculosis complex.

  18. Assessing Trace Evidence Left by Secure Deletion Programs

    NASA Astrophysics Data System (ADS)

    Burke, Paul; Craiger, Philip

    Secure deletion programs purport to permanently erase files from digital media. These programs are used by businesses and individuals to remove sensitive information from media, and by criminals to remove evidence of the tools or fruits of illegal activities. This paper focuses on the trace evidence left by secure deletion programs. In particular, five Windows-based secure deletion programs are tested to determine if they leave identifiable signatures after deleting a file. The results show that the majority of the programs leave identifiable signatures. Moreover, some of the programs do not completely erase file metadata, which enables forensic investigators to extract the name, size, creation date and deletion date of the "deleted" files.

  19. Tracking the assembly pathway of human immunodeficiency virus type 1 Gag deletion mutants by immunogold labeling.

    PubMed

    Wang, J J; Sandefur, S; Spearman, P; Chiou, C T; Chiang, P H; Ratner, L

    2001-12-01

    The Pr55gag gene product of human immunodeficiency virus type 1 (HIV-1) is sufficient to direct the formation of retrovirus-like particles (RVLPs). Recent biochemical evidence has indicated the presence of Gag intermediates in the cytoplasm; however, the Gag assembly process into RVLPs remains incompletely defined. The authors present here the subcellular localization of Gag mutant proteins in BSC40 and Jurkat cells by immunoelectron microscopy (IEM). The full Gag/Pol and Gag precursors, a C-terminal deletion mutant lacking a portion of nucleocapsid (NC), and all p6Gag gave rise to similar levels of RVLPs at the cell surface. A C-terminal deletion of all NC and p6Gag abrogated particle formation, whereas p24 was found in patches at the cell surface. Deletion of matrix (MA) sequences from Gag resulted in intracellular particles, and myristylation was not required for particle formation in the context of the MA deletion. Matrix expression was enhanced with Gag/Pol or Env coexpression as determined by semiquantitative IEM. p24 protein was targeted at vacuolar and mitochondrial membranes, but not at Golgi cisternae. In addition, aggregations of Gag intermediates and RVLPs in the cytoplasm, rough endoplasmic reticulum, cisternae, and mitochondria were noted. These results provide defined in situ evidence that HIV-1 particle assembly occurs in the cytosol in addition to budding at most intracellular membranes.

  20. FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy

    PubMed Central

    Shang, Na; Arteaga, Maribel; Chitsike, Lennox; Wang, Fang; Viswakarma, Navin; Breslin, Peter; Qiu, Wei

    2016-01-01

    Understanding the molecular mechanisms of liver regeneration is essential to improve the survival rate of patients after surgical resection of large amounts of liver tissue. Focal adhesion kinase (FAK) regulates different cellular functions, including cell survival, proliferation and cell migration. The role of FAK in liver regeneration remains unknown. In this study, we found that Fak is activated and induced during liver regeneration after two-thirds partial hepatectomy (PHx). We used mice with liver-specific deletion of Fak and investigated the role of Fak in liver regeneration in 2/3 PHx model (removal of 2/3 of the liver). We found that specific deletion of Fak accelerates liver regeneration. Fak deletion enhances hepatocyte proliferation prior to day 3 post-PHx but attenuates hepatocyte proliferation 3 days after PHx. Moreover, we demonstrated that the deletion of Fak in liver transiently increases EGFR activation by regulating the TNFα/HB-EGF axis during liver regeneration. Furthermore, we found more apoptosis in Fak-deficient mouse livers compared to WT mouse livers after PHx. Conclusion: Our data suggest that Fak is involved in the process of liver regeneration, and inhibition of FAK may be a promising strategy to accelerate liver regeneration in recipients after liver transplantation. PMID:27677358

  1. Deletions of the elastin gene in Williams Syndrome

    SciTech Connect

    Greenberg, F.; Nickerson, E.; McCaskill, C.

    1994-09-01

    To investigate deletions in the elastin gene in patients with Williams Syndrome (WS), we screened 37 patients and their parents for deletions in the elastin gene by both fluorescence in situ hybridization (FISH) using cosmid cELN272 containing the 5{prime} end of the elastin gene and by polymerase chain reaction (PCR) using a primer pair which amplifies intron 17 in the elastin gene, producing a polymorphic amplification product. Thirty-two patients have been investigated by both the FISH and PCR techniques, one patient was studied only by PCR, and 4 patients were studied only by FISH. Overall, 34 of 37 patients (92%) were deleted for the elastin gene. Using the PCR marker, 14 patients were informative and 12 were shown to be deleted [maternal (n=5) and paternal (n=7)]. Using cosmid cELN272, 33 of 36 patients demonstrated a deletion of chromosome 7q11.23. In one family, both the mother and daughter were deleted due to an apparently de novo deletion arising in the mother. Three patients were not deleted using the elastin cosmid; 2 of these patients have classic WS. Another non-deleted patient has the typical facial features and hypercalcemia but normal intelligence. These three patients will be important in delineating the critical region(s) responsible for the facial features, hypercalcemia, mental retardation and supravalvular aortic stenosis (SVAS). There was not an absolute correlation between deletions in elastin and SVAS, although these individuals may be at risk for other cardiovascular complications such as hypertention. Since the majority of WS patients are deleted for a portion of the elastin gene, most likely this marker will be an important diagnostic tool, although more patients will need to be studied. Those patients who are not deleted but clinically have WS will be missed using only this one marker. Expansion of the critical region to other loci and identification of additional markers will be essential for identifying all patients with WS.

  2. NPL deletion policy for RCRA-regulated TSD facilities finalized

    SciTech Connect

    1995-05-01

    Under a new policy published by EPA on March 20, 1995, certain sites may be deleted from the National Priorities List (NPL) and deferred to RCRA corrective action. To be deleted from the NPL, a site must (1) be regulated under RCRA as a treatment, storage, or disposal (TSD) facility and (2) meet the four criteria specified by EPA. The new NPL deletion policy, which does not pertain to federal TSD facilities, became effective on April 19, 1995. 1 tab.

  3. Analysis of partial AZFc deletions in Malaysian infertile male subjects.

    PubMed

    Almeamar, Hussein Ali; Ramachandran, Vasudevan; Ismail, Patimah; Nadkarni, Prashan; Fawzi, Nora

    2013-04-01

    Complete deletions in the AZF (a, b, and c) sub-regions of the Y-chromosome have been shown to contribute to unexplained male infertility. However, the role of partial AZFc deletions in male infertility remains to be verified. Three types of partial AZFc deletions have been identified. They are gr/gr, b1/b3, and b2/b3 deletions. A recent meta-analysis showed that ethnic and geographical factors might contribute to the association of partial AZFc deletions with male infertility. This study analyzed the association of partial AZFc deletions in Malaysian infertile males. Fifty two oligozoospermic infertile males and 63 fertile controls were recruited to this study. Screening for partial AZFc deletions was done using the two sequence-tagged sites approach (SY1291 and SY1191) which were analyzed using both the conventional PCR gel-electrophoresis and the high resolution melt, HRM method. Gr/gr deletions were found in 11.53% of the cases and 9.52% of the controls (p = 0.725). A B2/b3 deletion was found in one of the cases (p = 0.269). No B1/b3 deletions were identified in this study. The results of HRM analysis were consistent with those obtained using the conventional PCR gel-electrophoresis method. The HRM analysis was highly repeatable (95% limit of agreement was -0.0879 to 0.0871 for SY1191 melting temperature readings). In conclusion, our study showed that partial AZFc deletions were not associated with male infertility in Malaysian subjects. HRM analysis was a reliable, repeatable, fast, cost-effective, and semi-automated method which can be used for screening of partial AZFc deletions.

  4. Hereditary hemorrhagic telangiectasia: two distinct ENG deletions in one family.

    PubMed

    Wooderchak, W; Gedge, F; McDonald, M; Krautscheid, P; Wang, X; Malkiewicz, J; Bukjiok, C J; Lewis, T; Bayrak-Toydemir, P

    2010-11-01

    Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by aberrant vascular development. Mutations in endoglin (ENG) or activin A receptor type II-like 1 (ACVRL1) account for around 90% of HHT patients, 10% of those are large deletions or duplications. We report here the first observation of two distinct, large ENG deletions segregating in one pedigree. An ENG exon 4-7 deletion was observed in a patient with HHT. This deletion was identified in several affected family members. However, some affected family members had an ENG exon 3 deletion instead. These deletions were detected by multiplex ligation-dependent probe amplification and confirmed by mRNA sequencing and an oligo-CGH array. Linkage analysis revealed that one individual with the exon 3 deletion inherited the same chromosome from his mother who has the exon 4-7 deletion. This finding has important clinical implications because it shows that targeted family-specific mutation analysis for exon deletions could have led to the misdiagnosis of some affected family members. © 2010 John Wiley & Sons A/S.

  5. Molecular mapping within the mouse albino-deletion complex.

    PubMed

    Johnson, D K; Hand, R E; Rinchik, E M

    1989-11-01

    Induced germ-line deletion mutations in the mouse provide a malleable experimental system for in-depth molecular and functional analysis of large segments of the mammalian genome. To obtain an initial bank of molecular probes for the region of mouse chromosome 7 associated with the albino-deletion complex, random anonymous DNA clones, derived from a library constructed from flow-sorted chromosomes, were screened on DNAs from Mus musculus-Mus spretus F1 hybrids carrying large, multilocus, lethal albino deletions. Clones falling within a given deletion interval can easily be recognized because hybridization bands that represent restriction fragment length polymorphisms specific for the mutant (deleted) chromosome inherited from the M. musculus parent will be absent. Among 72 informative clones used as probes, one, which defines the locus D7OR1, mapped within two deletions that are 6-11 centimorgans in length. Submapping of this anonymous clone across a panel of 27 smaller deletions localized D7OR1 distal to a chromosomal subregion important for survival of the preimplantation embryo, proximal to globin [beta-chain (Hbb)], and near the shaker-1 (sh-1) locus. The results of these deletion-mapping experiments were also confirmed by standard three-point linkage analysis. This strategy for selection and rapid mapping of anonymous DNA probes to chromosomal segments corresponding to germ-line deletion mutations should contribute to the generation of more detailed physical and functional maps of genomic regions associated with mutant developmental phenotypes.

  6. Comprehensive Analysis of Pathogenic Deletion Variants in Fanconi Anemia Genes

    PubMed Central

    Flynn, Elizabeth K.; Kamat, Aparna; Lach, Francis P.; Donovan, Frank X.; Kimble, Danielle C.; Narisu, Narisu; Sanborn, Erica; Boulad, Farid; Davies, Stella M.; Gillio, Alfred P.; Harris, Richard E.; MacMillan, Margaret L.; Wagner, John E.; Smogorzewska, Agata; Auerbach, Arleen D.; Ostrander, Elaine A.; Chandrasekharappa, Settara C.

    2014-01-01

    Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution Comparative Genome Hybridization arrays (arrayCGH), Single Nucleotide Polymorphism arrays (SNParrays) and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by Non-Allelic Homologous Recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants. PMID:25168418

  7. Large-scale mitochondrial DNA deletions in skeletal muscle of patients with end-stage renal disease.

    PubMed

    Lim, P S; Cheng, Y M; Wei, Y H

    2000-09-01

    End-stage renal disease (ESRD) is associated with enhanced oxidative stress. This disease state provides a unique system for investigating the deleterious effect of exogenous sources of free radicals and reactive oxygen species (ROS) on mitochondrial DNA (mtDNA). To test the hypothesis that uremic milieu might cause more severe damage to mtDNA, we investigated the prevalence and abundance of mtDNA deletions in the skeletal muscles of ESRD patients. The results showed that the frequencies of occurrence of the 4977 bp and 7436 bp deletions of mtDNA in the muscle tissues of the older ESRD patients were higher than those of the younger patients. The frequency of occurrence of the 4977 bp-deleted mtDNA in the muscle was 33.3% for the patients in the age group of < 40 years, 66.6% in the 41-60-year-old group, 100% in the 61-80-year-old group, and 100% in patients >80 years of age, respectively. Only 22% of the normal aged controls carried the 4977 bp mtDNA deletion, whereas 77% (17/22) of the ESRD patients exhibited the mtDNA deletion. Using a semiquantitative PCR method, we determined the proportion of the 4977 bp-deleted mtDNA from the muscles that had been confirmed to harbor the deletion. We found that the proportions of the 4977 bp-deleted mtDNA in the muscle were significantly higher than those of the aged matched controls. Using long-range PCR techniques, a distinctive array of mtDNA deletions was demonstrated in the muscle of uremic patients. In summary, we found diverse and multiple mtDNA deletions in the skeletal muscles of ESRD patients. These deletions are more prevalent and abundant in ESRD patients than those found in normal populations. Accumulation of uremic toxins and impaired free radical scavenging systems may be responsible for the increased oxidative stress in ESRD patients. Such stress may result in oxidative damage and aging-associated mutation of the mitochondrial genome.

  8. ADRA2B deletion variant influences time-dependent effects of pre-learning stress on long-term memory.

    PubMed

    Zoladz, Phillip R; Dailey, Alison M; Nagle, Hannah E; Fiely, Miranda K; Mosley, Brianne E; Brown, Callie M; Duffy, Tessa J; Scharf, Amanda R; Earley, McKenna B; Rorabaugh, Boyd R

    2017-02-22

    Extensive work over the past few decades has shown that certain genetic variations interact with life events to confer increased susceptibility for the development of psychological disorders. The deletion variant of the ADRA2B gene, which has been associated with enhanced emotional memory and heightened amygdala responses to emotional stimuli, might confer increased susceptibility for the development of post-traumatic stress disorder (PTSD) or related phenotypes by increasing the likelihood of traumatic memory formation. Thus, we examined whether this genetic variant would predict stress effects on learning and memory in a non-clinical sample. Two hundred and thirty-five individuals were exposed to the socially evaluated cold pressor test or a control condition immediately or 30min prior to learning a list of words that varied in emotional valence and arousal level. Participants' memory for the words was tested immediately (recall) and 24h after learning (recall and recognition), and saliva samples were collected to genotype participants for the ADRA2B deletion variant. Results showed that stress administered immediately before learning selectively enhanced long-term recall in deletion carriers. Stress administered 30min before learning impaired recognition memory in male deletion carriers, while enhancing recognition memory in female deletion carriers. These findings provide additional evidence to support the idea that ADRA2B deletion variant carriers retain a sensitized stress response system, which results in amplified effects of stress on learning and memory. The accumulating evidence regarding this genetic variant implicates it as a susceptibility factor for traumatic memory formation and PTSD-related phenotypes.

  9. Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

    PubMed

    Chakrabarti, S; Joffe, S; Seidman, M M

    1985-09-01

    Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.

  10. Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

    PubMed Central

    Chakrabarti, S; Joffe, S; Seidman, M M

    1985-01-01

    Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools. Images PMID:3869955

  11. Adult-onset deletion of Pten increases islet mass and beta cell proliferation in mice

    PubMed Central

    Yang, Kai-Ting; Bayan, Jennifer-Ann; Zeng, Ni; Aggarwal, Richa; He, Lina; Peng, Zhechu; Kassa, Anketse; Kim, Melissa; Luo, Zhiou; Shi, Zhenrong; Medina, Vivian; Boddupally, Keerthi; Stiles, Bangyan L.

    2013-01-01

    Aims/hypothesis Adult beta cells have a diminished ability to proliferate. Phosphatase and tensin homologue (PTEN) is a lipid phosphatase that antagonises the function of the mitogenic phosphatidylinositol 3-kinase (PI3K) pathway. The objective of this study was to understand the role of PTEN and PI3K signalling in the maintenance of beta cells postnatally. Methods We developed a Ptenlox/lox; Rosa26lacZ; RIP-CreER+ model that permitted us to induce Pten deletion by treatment with tamoxifen in mature animals. We evaluated islet mass and function as well as beta cell proliferation in 3- and 12-month-old mice treated with tamoxifen (Pten deleted) vs mice treated with vehicle (Pten control). Results Deletion of Pten in juvenile (3-month-old) beta cells significantly induced their proliferation and increased islet mass. The expansion of islet mass occurred concomitantly with the enhanced ability of the Pten-deleted mice to maintain euglycaemia in response to streptozotocin treatment. In older mice (>12 months of age), deletion of Pten similarly increased islet mass and beta cell proliferation. This novel finding suggests that PTEN-regulated mechanisms may override the age-onset diminished ability of beta cells to respond to mitogenic stimulation. We also found that proteins regulating G1/S cell-cycle transition, such as cyclin D1, cyclin D2, p27 and p16, were altered when PTEN was lost, suggesting that they may play a role in PTEN/PI3K-regulated beta cell proliferation in adult tissue. Conclusions/interpretation The signals regulated by the PTEN/PI3K pathway are important for postnatal maintenance of beta cells and regulation of their proliferation in adult tissues. PMID:24162585

  12. Deletion of 6q16-q21 in human lymphoid malignancies: a mapping and deletion analysis.

    PubMed

    Jackson, A; Carrara, P; Duke, V; Sinclair, P; Papaioannou, M; Harrison, C J; Foroni, L

    2000-06-01

    Two distinct regions of minimal deletion (RMD) have been identified at 6q25-q27 in non-Hodgkin's lymphoma (RMD-1), and at 6q21-q23 in acute lymphoblastic leukemia (ALL; RMD-2) by loss of heterozygosity and fluorescence in situ hybridization studies. In this study, 30 overlapping yeast artificial chromosomes (YACs), 1 expressed sequence tag, and 11 novel YAC ends were identified using bidirectional YAC walks between markers D6S447 (proximal) and D6S246 (distal) in RMD-2. The genes AF6q21, human homologue of the Drosophila tailless (HTLX), CD24 antigen, the Kruppel-like zinc finger BLIMP1, and cyclin C (CCNC), previously mapped to 6q21, were accurately positioned in a telomere-to-centromere orientation. Approximately 3.5 Mb were found to separate the BLIMP1 (adjacent to D6S447) and AF6q21 genes (telomeric to D6S246). Deletions of 6q were investigated in 21 cases of ALL using the newly characterized YAC clones in dual-color fluorescence in situ hybridization studies. A region centromeric to D6S447 (containing marker D6S283) and a region telomeric to marker CHLC.GGAT16CO2 (and containing marker D6S268) were identified as distinct and nonoverlapping regions of deletion in ALL.

  13. Transcriptional enhancer from milk protein genes

    DOEpatents

    Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

    1999-01-01

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  14. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, G.F.; Schmidhauser, C.T.; Bissell, M.J.

    1999-12-21

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  15. Orbital myeloid sarcoma in an adult with acute myeloid leukemia, FAB M1, and 12p-deletion.

    PubMed

    Ple-plakon, Patricia A; Demirci, Hakan; Cheng, Jason X; Elner, Victor M

    2013-01-01

    A 49-year-old woman with acute myeloid leukemia, FAB M1 subtype, and 12p deletion, presented with progressive right proptosis and diplopia for 1 week. Orbital CT revealed a homogenously enhancing, orbital mass engulfing the inferior rectus muscle. Histopathology revealed myeloid sarcoma for which she underwent external beam radiotherapy. Subsequently, there was no sign of local recurrence, but she succumbed to leukemia involving the central nervous system. This is the first case, to the authors' knowledge, of an orbital sarcoma of FAB M1 myeloblasts bearing a 12p deletion.

  16. Using Topic Modeling and Text Embeddings to Predict Deleted Tweets

    SciTech Connect

    Potash, Peter J.; Bell, Eric B.; Harrison, Joshua J.

    2016-02-29

    Predictive models for tweet deletion have been a relatively unexplored area of Twitter-related computational research. We first approach the deletion of tweets as a spam detection problem, applying a small set of handcrafted features to improve upon the current state-of-the- art in predicting deleted tweets. Next, we apply our approach to a dataset of deleted tweets that better reflects the current deletion rate. Since tweets are deleted for reasons beyond just the presence of spam, we apply topic modeling and text embeddings in order to capture the semantic content of tweets that can lead to tweet deletion. Our goal is to create an effective model that has a low-dimensional feature space and is also language-independent. A lean model would be computationally advantageous processing high-volumes of Twitter data, which can reach 9,885 tweets per second. Our results show that a small set of spam-related features combined with word topics and character-level text embeddings provide the best f1 when trained with a random forest model. The highest precision of the deleted tweet class is achieved by a modification of paragraph2vec to capture author identity.

  17. 29 CFR 1610.20 - Deletion of exempted matters.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 4 2010-07-01 2010-07-01 false Deletion of exempted matters. 1610.20 Section 1610.20 Labor... Production or Disclosure Under 5 U.S.C. 552 § 1610.20 Deletion of exempted matters. Where requested records contain matters which are exempted under 5 U.S.C. 552(b) but which matters are reasonably segregable...

  18. 29 CFR 1610.20 - Deletion of exempted matters.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 4 2011-07-01 2011-07-01 false Deletion of exempted matters. 1610.20 Section 1610.20 Labor... Production or Disclosure Under 5 U.S.C. 552 § 1610.20 Deletion of exempted matters. Where requested records contain matters which are exempted under 5 U.S.C. 552(b) but which matters are reasonably segregable...

  19. Linguistic and Psychomotor Development in Children with Chromosome 14 Deletions

    ERIC Educational Resources Information Center

    Zampini, Laura; D'Odorico, Laura; Zanchi, Paola; Zollino, Marcella; Neri, Giovanni

    2012-01-01

    The present study focussed on a specific type of rare genetic condition: chromosome 14 deletions. Children with this genetic condition often show developmental delays and brain and neurological problems, although the type and severity of symptoms varies depending on the size and location of the deleted genetic material. The specific aim of the…

  20. Multivariate Variable Deletion Methods: Don't Do Stepwise

    ERIC Educational Resources Information Center

    Kadhi, TauGamba

    2003-01-01

    This paper explains the theory and methodology behind the use of variable deletion in canonical correlational analysis (CCA). Both the Capraro and Capraro (2002) and the Cantrell (1997) data tables are evaluated and explained in order to clarify strategies utilized. Understanding of variable deletion strategies and their proper usages in a CCA…

  1. Towards earlier diagnosis of 22q11 deletions

    PubMed Central

    Tobias, E; Morrison, N; Whiteford, M; Tolmie, J

    1999-01-01

    Over a 7 year period, 551 patients were investigated for the presence of a chromosome 22q11 deletion by fluorescence in situ hybridisation. Analysis of the presenting features of the 67 individuals with this chromosome deletion permitted us to devise guidelines to facilitate early diagnosis.

 PMID:10569971

  2. A Note On Deletion Rules in Fast Speech.

    ERIC Educational Resources Information Center

    Hewlett, Nigel

    In fast speech, certain segments pronounced in careful speech may be deleted. Rules of a generative phonology have been used to account for fast speech forms. An alternative approach is suggested which views fast speech deletions as merely limiting cases of segment reduction, under conditions of increased tempo and/or casualness. To complement…

  3. 75 FR 60739 - Procurement List; Additions and Deletions

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  4. 19 CFR 142.49 - Deletion of C-4 Code.

    Code of Federal Regulations, 2011 CFR

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    Code of Federal Regulations, 2012 CFR

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  6. 37 CFR 2.35 - Adding, deleting, or substituting bases.

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    Code of Federal Regulations, 2011 CFR

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  8. 37 CFR 2.35 - Adding, deleting, or substituting bases.

    Code of Federal Regulations, 2014 CFR

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  9. 78 FR 53733 - Procurement List Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

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    Federal Register 2010, 2011, 2012, 2013, 2014

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  11. Coexistence of 9p Deletion Syndrome and Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Günes, Serkan; Ekinci, Özalp; Ekinci, Nuran; Toros, Fevziye

    2017-01-01

    Deletion or duplication of the short arm of chromosome 9 may lead to a variety of clinical conditions including craniofacial and limb abnormalities, skeletal malformations, mental retardation, and autism spectrum disorder. Here, we present a case report of 5-year-old boy with 9p deletion syndrome and autism spectrum disorder.

  12. Linguistic and Psychomotor Development in Children with Chromosome 14 Deletions

    ERIC Educational Resources Information Center

    Zampini, Laura; D'Odorico, Laura; Zanchi, Paola; Zollino, Marcella; Neri, Giovanni

    2012-01-01

    The present study focussed on a specific type of rare genetic condition: chromosome 14 deletions. Children with this genetic condition often show developmental delays and brain and neurological problems, although the type and severity of symptoms varies depending on the size and location of the deleted genetic material. The specific aim of the…

  13. Computed structures of point deletion mutants and their enzymatic activities

    PubMed Central

    Berrondo, Monica; Gray, Jeffrey J.

    2011-01-01

    Point deletions in enzymes can vary in effect from negligible to complete loss of activity, however, these effects are not generally predictable. Deletions are widely observed in nature and often result in diseases such as cancer, cystic fibrosis, or osteogenesis imperfecta. Here, we have developed an algorithm to model the perturbed structures of deletion mutants with the ultimate goal of predicting their activities. The algorithm works by deleting the specified residue from the wild-type structure, creating a gap that is closed using a combination of local and global moves that change the backbone torsion angles of the protein structure. On a set of five proteins for which both wild-type and deletion mutant x-ray crystal structures are available, the algorithm produces deep, narrow energy funnels within 1.5 Å of the crystal structure for the deletion mutants. To assess the ability of our algorithm to predict activity from the predicted structures, we tested the correlation of experimental activity with several measures of the predicted structure ensemble using a set of 45 point deletions from ricin. Estimates incorporating likely prevalence of active and inactive deletion sites suggest that activity can be predicted correctly over 60% of the time from the active site rmsd of the lowest energy predicted structures. The predictions are stronger than simple sequence organization measures, but more fundamental work is required in structure prediction and enzyme activity determination to allow consistent prediction of activity. PMID:21905110

  14. Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

    PubMed Central

    Calì, Francesco; Ruggeri, Giuseppa; Vinci, Mirella; Meli, Concetta; Carducci, Carla; Leuzzi, Vincenzo; Pozzessere, Simone; Schinocca, Pietro; Ragalmuto, Alda; Chiavetta, Valeria; Miccichè, Salvatore

    2010-01-01

    A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics. PMID:19946181

  15. 77 FR 12816 - Procurement List Proposed Addition and Deletions

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  16. Coexistence of 9p Deletion Syndrome and Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Günes, Serkan; Ekinci, Özalp; Ekinci, Nuran; Toros, Fevziye

    2017-01-01

    Deletion or duplication of the short arm of chromosome 9 may lead to a variety of clinical conditions including craniofacial and limb abnormalities, skeletal malformations, mental retardation, and autism spectrum disorder. Here, we present a case report of 5-year-old boy with 9p deletion syndrome and autism spectrum disorder.

  17. 75 FR 49481 - Procurement List; Additions and Deletion

    Federal Register 2010, 2011, 2012, 2013, 2014

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  18. 75 FR 56995 - Procurement List Proposed Additions and Deletion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-17

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  19. 44 CFR 5.27 - Deletion of identifying details.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 44 Emergency Management and Assistance 1 2010-10-01 2010-10-01 false Deletion of identifying details. 5.27 Section 5.27 Emergency Management and Assistance FEDERAL EMERGENCY MANAGEMENT AGENCY... Availability of General Agency Information, Rules, Orders, Policies, and Similar Material § 5.27 Deletion of...

  20. Chromosome 7 monosomy and deletions in myeloproliferative diseases.

    PubMed

    Tripputi, P; Cassani, B; Alfano, R; Graziani, D; Cigognini, D; Doi, P; Bignotto, M; Corneo, G; Coggi, G

    2001-09-01

    We studied deletion and monosomy of chromosome 7 in 150 patients with myeloproliferative diseases. We found 8/150 patients with monosomy 7 by cytogenetics and 4/150 with deletions of the long arm of chromosome 7 by restriction fragment length polymorphism (RFLP) analysis performed with Southern and polymerase chain reaction. To overcome limitation of RFLP analysis, we restricted loss of heterozygosity study with microsatellites to 45 patients, observing deletion 7q31.1 in 7/45 patients. In all patients with molecular alterations the deletion was observed only in myeloid cells, while the monosomy was detected in both myeloid precursor and lymphocytes. This finding suggests a CD34-totipotent stem cell origin for the monosomy and a colony forming unit - granulocyte, erythrocyte, monocyte, megakaryocytes (CFU-GEMM) stem cell origin for the deletions.

  1. Delineation of 14q32.3 deletion syndrome.

    PubMed

    Ortigas, A P; Stein, C K; Thomson, L L; Hoo, J J

    1997-06-01

    A patient with a 14q32.3 terminal band deletion and cat cry is reported. Review of four other 14q32.3 deletion cases suggests the possible presence of a recognisable 14q32.3 terminal deletion syndrome, which is characterised by (1) apparently postnatal onset of small head size in comparison to body size, (2) high forehead with lateral hypertrichosis, (3) epicanthic folds, (4) broad nasal bridge, (5) high arched palate, (6) single palmar crease, and (7) mild to moderate developmental delay. Although none of the above seven features in unique to this syndrome, and indeed are quite common in other chromosomal disorders or genetic syndromes, patients with a terminal 14q32.3 deletion do show a recognisable facial gestalt. Interestingly, unlike ring chromosome 14, the 14q32.3 terminal deletion has rarely been reported, possibly because it is harder to detect, and an optimal chromosome preparation is required for its identification.

  2. Attenuation of monkeypox virus by deletion of genomic regions

    USGS Publications Warehouse

    Lopera, Juan G.; Falendysz, Elizabeth A.; Rocke, Tonie E.; Osorio, Jorge E.

    2015-01-01

    Monkeypox virus (MPXV) is an emerging pathogen from Africa that causes disease similar to smallpox. Two clades with different geographic distributions and virulence have been described. Here, we utilized bioinformatic tools to identify genomic regions in MPXV containing multiple virulence genes and explored their roles in pathogenicity; two selected regions were then deleted singularly or in combination. In vitro and in vivostudies indicated that these regions play a significant role in MPXV replication, tissue spread, and mortality in mice. Interestingly, while deletion of either region led to decreased virulence in mice, one region had no effect on in vitro replication. Deletion of both regions simultaneously also reduced cell culture replication and significantly increased the attenuation in vivo over either single deletion. Attenuated MPXV with genomic deletions present a safe and efficacious tool in the study of MPX pathogenesis and in the identification of genetic factors associated with virulence.

  3. Growth patterns of patients with 1p36 deletion syndrome.

    PubMed

    Sangu, Noriko; Shimojima, Keiko; Shimada, Shino; Ando, Tomohiro; Yamamoto, Toshiyuki

    2014-05-01

    1p36 deletion syndrome is one of the most common subtelomeric deletion syndromes. Obesity is frequently observed in patients with this syndrome. Thus, it is important to evaluate the growth status of an individual patient. For this purpose, we accumulated recorded growth data from 44 patients with this syndrome and investigated the growth patterns of patients. Most of the patients showed weight parameters within normal limits, whereas a few of these patients showed intrauterine growth delay and microcephaly. The length of the patients after birth was under the 50th centile in most patients. Many patients showed poor weight gain after birth, and only two female patients were overweight. These findings indicate two different phenotypes of the 1p36 deletion syndrome. The overweight patients with 1p36 deletion started excessive weight gain after two years of life. This characteristic of the patients with 1p36 deletion syndrome is similar to Prader-Willi syndrome.

  4. Specific mitochondrial DNA deletions in idiopathic dilated cardiomyopathy.

    PubMed

    Marin-Garcia, J; Goldenthal, M J; Ananthakrishnan, R; Pierpont, M E; Fricker, F J; Lipshultz, S E; Perez-Atayde, A

    1996-02-01

    Structural changes in human mitochondrial DNA (mtDNA) have been implicated in a number of clinical conditions with dysfunctions in oxidative phosphorylation called OX-PHOS diseases, some of which have cardiac involvement. The objective of this study was to assess the frequency and extent of specific mitochondrial DNA deletions in idiopathic dilated cardiomyopathy. DNA extracted from tissue derived from the left ventricle of 41 patients with idiopathic dilated cardiomyopathy and 17 controls was amplified by polymerase chain reaction using specific primers to assess the incidence and proportion of 5-kb and 7.4-kb deletions in mitochondrial DNA. In reactions using primers to detect the 5-kb deletion, an amplified product of 593 bp was found in low abundance relative to undeleted mitochondrial DNA but with high frequency in a number of controls and patients. A second deletion of 7.4 kb in size was also frequently present in controls and patients. In contrast to previous reports, these deletions were found to be present in both controls and in cardiomyopathic patients, 18 years and younger, including several infants. The 7.4-kb deletion was prominently increased in both frequency and in its proportion relative to undeleted mitochondrial DNA in patients 40 years and older with idiopathic dilated cardiomyopathy. At variance with current literature our study reports a significant presence of both 5 and 7.4-kb deletions in the young and a higher frequency and quantity of the 7.4-kb deletion in the older cardiomyopathic patients in comparison with controls. The increased accumulation of the 7.4-kb deletion as both a function of aging and cardiomyopathy is suggestive that this specific mitochondrial DNA deletion arises more likely as an effect of heart dysfunction rather than as a primary cause of cardiomyopathy.

  5. "Oops, I deleted it"--a solution for recovering deleted or reformatted digital images from memory cards.

    PubMed

    Revankar, Ameet V; Gandedkar, Narayan H; Ganeshkar, Sanjay V

    2009-06-01

    Capturing patient photographs on digital flash cards rather than film has several advantages, but also a few disadvantages. Among the latter, the primary disadvantage is the possibility of accidentally deleting or corruping the files before they are downloaded from the memory card. In this article, we describe utilities that enable image recovery from deleted, reformatted, or physically damaged flash memory cards.

  6. 'Deletion rescue' by mitotic 11q uniparental disomy in a family with recurrence of 11q deletion Jacobsen syndrome.

    PubMed

    Johnson, J P; Haag, M; Beischel, L; McCann, C; Phillips, S; Tunby, M; Hansen, J; Schwanke, C; Reynolds, J F

    2014-04-01

    We describe a family with recurrent 11q23-qter deletion Jacobsen syndrome in two affected brothers, with unique mosaic deletion 'rescue' through development of uniparental disomy (UPD) in the mother and one of the brothers. Inheritance studies show that the deleted chromosome is of maternal origin in both boys, and microarray shows a break near the ASAM gene. Parental lymphocyte chromosomes were normal. However, the mother is homozygous in lymphocytes for all loci within the deleted region in her sons, and presumably has UPD for this region. In addition, she is mosaic for the 11q deletion seen in her sons at a level of 20-30% in skin fibroblasts. We hypothesize that one of her #11 chromosomes shows fragility, that breakage at 11q23 occurred with telomeric loss in some cells, but 'rescue' from the deletion occurred in most cells by the development of mitotic UPD. She apparently carries the 11q deletion in her germ line resulting in recurrence of the syndrome. The older son is mosaic for the 11q cell line (70-88%, remainder 46,XY), and segmental UPD11 'rescue' apparently also occurred in his cytogenetically normal cells. This is a novel phenomenon restoring disomy to an individual with a chromosomal deletion.

  7. Improved molecular diagnosis of the common recurrent intragenic deletion mutation in IKBKG in a Filipino family with incontinentia pigmenti.

    PubMed

    Guevara, Bryan Edgar K; Hsu, Chao-Kai; Liu, Lu; Feast, Alice; Alabado, Karen Lee P; Lacuesta, Maricarr Pamela M; Lee, Julia Yu-Yun; McGrath, John A

    2016-05-01

    Incontinentia pigmenti is a rare, multisystem X-linked dominant genetic disorder caused by mutations in IKBKG, the encoding inhibitor of kappa light polypeptide gene enhancer in B-cells. Almost 80% of all cases result from a recurrent intragenic deletion mutation that removes exon 4-10. At present, this mutation can be detected by a multi-primer polymerase chain reaction (PCR) technique although current protocols may preferentially amplify the wild-type allele and miss the deletion. Here, we report a female infant with incontinentia pigmenti that also affected her mother and sister, and two spontaneously aborted male siblings. We developed a modified PCR amplification method that provides more robust detection of the exon 4-10 deletion mutation, which was demonstrated in all affected females in this pedigree.

  8. ACE insert/delete polymorphism and atherosclerosis.

    PubMed

    Scheer, W Douglas; Boudreau, Donald A; Hixson, James E; McGill, Henry C; Newman, William P; Tracy, Richard E; Zieske, Arthur W; Strong, Jack P

    2005-02-01

    We report on the results of a large autopsy study focusing upon the hypothesis that deletion of the Alu insert in the angiotensin converting enzyme (ACE) gene is associated with: (a) greater prevalence or extent of atherosclerosis in the aorta and coronary arteries; and (b) microscopic qualities of established atherosclerotic plaques in the coronary arteries. This study was conducted in young US black (n=290) and white (n=379) males using available materials and data from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, a multi-center cooperative autopsy study organized in 1985 to explore the relationships of known cardiovascular risk factors to atherosclerosis in victims of accidents, homicides, or suicides in the age range of 15-34 years. The results provide strong evidence that ACE genotype may not be a predictor of either the prevalence or the extent of the lesions of atherosclerosis in the right coronary artery or the aorta of young adults, an observation that confirms previous studies that estimated the prevalence and extent of atherosclerosis using coronary angiography. In addition, the results suggest that ACE genotype does not contribute to the formation of atherosclerotic lesions that have the characteristics of vulnerable plaques in the left anterior descending coronary artery of young adults.

  9. Tau deletion promotes brain insulin resistance

    PubMed Central

    Marciniak, Elodie; Leboucher, Antoine; Caron, Emilie; Ahmed, Tariq; Tailleux, Anne; Dumont, Julie; Issad, Tarik; Gerhardt, Ellen; Pagesy, Patrick; Vileno, Margaux; Hamdane, Malika; Bantubungi, Kadiombo; Lancel, Steve; Demeyer, Dominique; Eddarkaoui, Sabiha; Vallez, Emmanuelle; Vieau, Didier; Humez, Sandrine; Faivre, Emilie; Grenier-Boley, Benjamin; Outeiro, Tiago F.; Amouyel, Philippe; Balschun, Detlef

    2017-01-01

    The molecular pathways underlying tau pathology–induced synaptic/cognitive deficits and neurodegeneration are poorly understood. One prevalent hypothesis is that hyperphosphorylation, misfolding, and fibrillization of tau impair synaptic plasticity and cause degeneration. However, tau pathology may also result in the loss of specific physiological tau functions, which are largely unknown but could contribute to neuronal dysfunction. In the present study, we uncovered a novel function of tau in its ability to regulate brain insulin signaling. We found that tau deletion leads to an impaired hippocampal response to insulin, caused by altered IRS-1 and PTEN (phosphatase and tensin homologue on chromosome 10) activities. Our data also demonstrate that tau knockout mice exhibit an impaired hypothalamic anorexigenic effect of insulin that is associated with energy metabolism alterations. Consistently, we found that tau haplotypes are associated with glycemic traits in humans. The present data have far-reaching clinical implications and raise the hypothesis that pathophysiological tau loss-of-function favors brain insulin resistance, which is instrumental for cognitive and metabolic impairments in Alzheimer’s disease patients. PMID:28652303

  10. A highly penetrant form of childhood apraxia of speech due to deletion of 16p11.2.

    PubMed

    Fedorenko, Evelina; Morgan, Angela; Murray, Elizabeth; Cardinaux, Annie; Mei, Cristina; Tager-Flusberg, Helen; Fisher, Simon E; Kanwisher, Nancy

    2016-02-01

    Individuals with heterozygous 16p11.2 deletions reportedly suffer from a variety of difficulties with speech and language. Indeed, recent copy-number variant screens of children with childhood apraxia of speech (CAS), a specific and rare motor speech disorder, have identified three unrelated individuals with 16p11.2 deletions. However, the nature and prevalence of speech and language disorders in general, and CAS in particular, is unknown for individuals with 16p11.2 deletions. Here we took a genotype-first approach, conducting detailed and systematic characterization of speech abilities in a group of 11 unrelated children ascertained on the basis of 16p11.2 deletions. To obtain the most precise and replicable phenotyping, we included tasks that are highly diagnostic for CAS, and we tested children under the age of 18 years, an age group where CAS has been best characterized. Two individuals were largely nonverbal, preventing detailed speech analysis, whereas the remaining nine met the standard accepted diagnostic criteria for CAS. These results link 16p11.2 deletions to a highly penetrant form of CAS. Our findings underline the need for further precise characterization of speech and language profiles in larger groups of affected individuals, which will also enhance our understanding of how genetic pathways contribute to human communication disorders.

  11. Stress resistance and lifespan are increased in C. elegans but decreased in S. cerevisiae by mafr-1/maf1 deletion

    PubMed Central

    Cai, Ying; Wei, Yue-Hua

    2016-01-01

    Maf1 is a conserved effector of the mechanistic target of rapamycin (mTOR), an aging promoting kinase. However, whether Maf1 is required for lifespan extension caused by mTOR inhibition, such as dietary restriction (DR) or calorie restriction (CR) remains elusive. Here we show that deletion of maf1 in the budding yeast S. cerevisiae but not mafr-1 in C. elegans prevents DR or CR to extend lifespan. Interestingly, mafr-1 deletion increases stress tolerance and extends lifespan. MAFR-1 is phosphorylated in a mTOR-dependent manner and mafr-1 deletion alleviates the inhibition of tRNA synthesis caused by reduced mTOR activity. We find that the opposite effect of mafr-1 deletion on lifespan is due to an enhancement of stress response, including oxidative stress response, mitochondrial unfolded protein response (UPRmt) and autophagy. mafr-1 deletion also attenuates the paralysis of a C. elegans model of Alzheimer's disease. Our study reveals distinct mechanisms of lifespan regulation by Maf1 and MAFR-1. PMID:26934328

  12. Stress resistance and lifespan are increased in C. elegans but decreased in S. cerevisiae by mafr-1/maf1 deletion.

    PubMed

    Cai, Ying; Wei, Yue-Hua

    2016-03-08

    Maf1 is a conserved effector of the mechanistic target of rapamycin (mTOR), an aging promoting kinase. However, whether Maf1 is required for lifespan extension caused by mTOR inhibition, such as dietary restriction (DR) or calorie restriction (CR) remains elusive. Here we show that deletion of maf1 in the budding yeast S. cerevisiae but not mafr-1 in C. elegans prevents DR or CR to extend lifespan. Interestingly, mafr-1 deletion increases stress tolerance and extends lifespan. MAFR-1 is phosphorylated in a mTOR-dependent manner and mafr-1 deletion alleviates the inhibition of tRNA synthesis caused by reduced mTOR activity. We find that the opposite effect of mafr-1 deletion on lifespan is due to an enhancement of stress response, including oxidative stress response, mitochondrial unfolded protein response (UPRmt) and autophagy. mafr-1 deletion also attenuates the paralysis of a C. elegans model of Alzheimer's disease. Our study reveals distinct mechanisms of lifespan regulation by Maf1 and MAFR-1.

  13. The HLA-G 14-base pair deletion allele and the deletion/deletion genotype are associated with persistent HBe antigenemia in chronic hepatis B infection.

    PubMed

    Ferreira, Sandro da Costa; Chachá, Silvana Gama Florêncio; Souza, Fernanda Fernandes; Teixeira, Andreza Corrêa; Santana, Rodrigo de Carvalho; Deghaide, Neifi Hassan Saloun; Rodrigues, Sandra; Marano, Leonardo A; Mendes-Junior, Celso Teixeira; Ramalho, Leandra Naira Zambelli; Zucoloto, Sérgio; Donadi, Eduardo Antônio; Martinelli, Ana de Lourdes Candolo

    2017-02-01

    HLA-G has well-recognized immunomodulatory properties, and this molecule is frequently expressed in the livers of hepatitis B virus (HBV)-infected patients. Because the HLA-G 14 bp-insertion/deletion polymorphism (rs371194629) has been associated with the magnitude of HLA-G expression, we evaluated this polymorphism in the recognized evolutionary forms of chronic HBV infection. We studied 196 chronic HBV-infected patients (118 HBeAg-negative chronic hepatitis, 53 HBeAg-positive chronic hepatitis and 25 inactive carriers exhibiting low levels of serum HBVDNA and persistently normal ALT levels), and 202 healthy individuals. Chronic hepatitis HLA-G typing was performed using PCR-amplified DNA hybridized with specific primers. The frequencies of the insertion/deletion alleles and genotypes were very similar in patients and controls. After patient stratification according to the evolutionary form of the chronic HBV infection, the frequencies of the deletion allele (P=0.0460; OR=1.26; 95%CI=1.01-1.45) and of the deletion/deletion genotype (P=0.0356; OR=2.08; 95%CI=1.05-4.09) were overrepresented in HBeAg-positive patients when compared to HBeAg-negative patients. No differences were observed when HBV inactive carriers were compared to HBeAg-negative chronic hepatitis patients. Because the 14-bp deletion allele has been associated with increased HLA-G production and because HLA-G may down regulate the cytotoxic activity of TCD8 and NK cells, patients exhibiting the 14-bp deletion allele at single or double doses are at increased risk for developing chronic forms of HBV associated with persistent viremia and worse prognoses. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  14. Molecular characterization of patients with 18q23 deletions.

    PubMed Central

    Strathdee, G; Sutherland, R; Jonsson, J J; Sataloff, R; Kohonen-Corish, M; Grady, D; Overhauser, J

    1997-01-01

    The 18q- syndrome is a deletion syndrome that is characterized by mental retardation, hearing loss, midfacial hypoplasia, growth deficiency, and limb anomalies. Most patients with this syndrome have deletions from 18q21-qter. We report on three patients with deletions of 18q23. A mother and daughter with identical deletions of 18q23 have many of the typical features of the 18q- syndrome, including midfacial hypoplasia and hearing loss. In contrast, the third patient has few of the symptoms of the 18q- syndrome. A contig of the 18q23 region was generated to aid in the mapping of the breakpoints. FISH was used to map both breakpoints to the same YAC clone. Furthermore, somatic-cell hybrids from the daughter and the third patient were isolated. The mapping results of sequence-tagged sites relative to the two breakpoints were identical, suggesting that the two deletion breakpoints map very close to one another. The analyses of these patients demonstrate that the critical region for the 18q- syndrome maps to 18q23 but that a deletion of 18q23 does not always lead to the clinical features associated with the syndrome. These patients demonstrate the wide phenotypic variability associated with deletions of 18q. Images Figure 2 Figure 3 PMID:9106532

  15. Recurrent deletions of IKZF1 in pediatric acute myeloid leukemia

    PubMed Central

    de Rooij, Jasmijn D.E.; Beuling, Eva; van den Heuvel-Eibrink, Marry M.; Obulkasim, Askar; Baruchel, André; Trka, Jan; Reinhardt, Dirk; Sonneveld, Edwin; Gibson, Brenda E.S.; Pieters, Rob; Zimmermann, Martin; Zwaan, C. Michel; Fornerod, Maarten

    2015-01-01

    IKAROS family zinc finger 1/IKZF1 is a transcription factor important in lymphoid differentiation, and a known tumor suppressor in acute lymphoid leukemia. Recent studies suggest that IKZF1 is also involved in myeloid differentiation. To investigate whether IKZF1 deletions also play a role in pediatric acute myeloid leukemia, we screened a panel of pediatric acute myeloid leukemia samples for deletions of the IKZF1 locus using multiplex ligation-dependent probe amplification and for mutations using direct sequencing. Three patients were identified with a single amino acid variant without change of IKZF1 length. No frame-shift mutations were found. Out of 11 patients with an IKZF1 deletion, 8 samples revealed a complete loss of chromosome 7, and 3 cases a focal deletion of 0.1–0.9Mb. These deletions included the complete IKZF1 gene (n=2) or exons 1–4 (n=1), all leading to a loss of IKZF1 function. Interestingly, differentially expressed genes in monosomy 7 cases (n=8) when compared to non-deleted samples (n=247) significantly correlated with gene expression changes in focal IKZF1-deleted cases (n=3). Genes with increased expression included genes involved in myeloid cell self-renewal and cell cycle, and a significant portion of GATA target genes and GATA factors. Together, these results suggest that loss of IKZF1 is recurrent in pediatric acute myeloid leukemia and might be a determinant of oncogenesis in acute myeloid leukemia with monosomy 7 PMID:26069293

  16. Do listeners recover "deleted" final /t/ in German?

    PubMed

    Zimmerer, Frank; Reetz, Henning

    2014-01-01

    Reduction and deletion processes occur regularly in conversational speech. A segment that is affected by such reduction and deletion processes in many Germanic languages (e.g., Dutch, English, German) is /t/. There are similarities concerning the factors that influence the likelihood of final /t/ to get deleted, such as segmental context. However, speakers of different languages differ with respect to the acoustic cues they leave in the speech signal when they delete final /t/. German speakers usually lengthen a preceding /s/ when they delete final /t/. This article investigates to what extent German listeners are able to reconstruct /t/ when they are presented with fragments of words where final /t/ has been deleted. It aims also at investigating whether the strategies that are used by German depend on the length of /s/, and therefore whether listeners are using language-specific cues. Results of a forced-choice segment detection task suggest that listeners are able to reconstruct deleted final /t/ in about 45% of the times. The length of /s/ plays some role in the reconstruction, however, it does not explain the behavior of German listeners completely.

  17. Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation

    SciTech Connect

    Nussbaum, R.L.; Lesko, J.G.; Lewis, R.A.; Ledbetter, S.A.; Ledbetter, D.H.

    1987-09-01

    Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. The authors have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmed by high-resolution cytogenetic analysis. They used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.

  18. Impact of partial DAZ1/2 deletion and partial DAZ3/4 deletion on male infertility.

    PubMed

    Zhang, Yuening; Li, Muyan; Xiao, Feifan; Teng, Ruobing; Zhang, Chengdong; Lan, Aihua; Gu, Kailong; Li, Jiatong; Wang, Di; Li, Hongtao; Jiang, Li; Zeng, Siping; He, Min; Huang, Yi; Guo, Peifen; Zhang, Xinhua; Yang, Xiaoli

    2015-10-15

    This study aims to investigate the effect of the partial DAZ1/2 deletion and partial DAZ3/4 deletion on male infertility through a comprehensive literature search. All case-control studies related to partial DAZ1/2 and DAZ3/4 deletions and male infertility risk were included in our study. Odd ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association and its precision, respectively. Eleven partial DAZ1/2 deletion and nine partial DAZ3/4 deletion studies were included. Partial DAZ1/2 deletion was significantly associated with male infertility risk in the overall analysis (ORs=2.58, 95%CI: 1.60-4.18, I(2)=62.1%). Moreover, in the subgroup analysis stratified by ethnicity, partial DAZ1/2 deletion was significantly associated with male infertility risk in the East Asian populations under the random effect model (ORs=2.96, 95%CI: 1.87-4.71, I(2)=51.3%). Meanwhile, the analysis suggested that partial DAZ3/4 deletion was not associated with male infertility risk in East-Asian ethnicity (ORs=1.02, 95%CI: 0.54-1.92, I(2)=71.3%), but not in Non-East Asian under the random effect model (ORs=3.56, 95%CI: 1.13-11.23, I(2)=0.0%,). More interestingly, partial DAZ1/2 deletion was associated with azoospermia (ORs=2.63, 95%CI: 1.19-5.81, I(2)=64.7%) and oligozoospermia (ORs=2.53, 95%CI: 1.40-4.57, I(2)=51.8%), but partial DAZ3/4 deletion was not associated with azoospermia (ORs=0.71, 95%CI: 0.23-2.22, I(2)=71.7%,) and oligozoospermia (ORs=1.21, 95%CI: 0.65-2.24, I(2)=55.5%). In our meta-analysis, partial DAZ1/2 deletion is a risk factor for male infertility and different ethnicities have different influences, whereas partial DAZ3/4 deletion has no effect on fertility but partial DAZ3/4 deletion might have an impact on Non-East Asian male.

  19. Detection of genomic deletions in rice using oligonucleotide microarrays

    PubMed Central

    Bruce, Myron; Hess, Ann; Bai, Jianfa; Mauleon, Ramil; Diaz, M Genaleen; Sugiyama, Nobuko; Bordeos, Alicia; Wang, Guo-Liang; Leung, Hei; Leach, Jan E

    2009-01-01

    Background The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL). However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. Results We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip® allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to ~500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations . Conclusion We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a database saturated with deletions across the rice genome. This community resource can continue

  20. Performance of quantum cloning and deleting machines over coherence

    NASA Astrophysics Data System (ADS)

    Karmakar, Sumana; Sen, Ajoy; Sarkar, Debasis

    2017-10-01

    Coherence, being at the heart of interference phenomena, is found to be an useful resource in quantum information theory. Here we want to understand quantum coherence under the combination of two fundamentally dual processes, viz., cloning and deleting. We found the role of quantum cloning and deletion machines with the consumption and generation of quantum coherence. We establish cloning as a cohering process and deletion as a decohering process. Fidelity of the process will be shown to have connection with coherence generation and consumption of the processes.

  1. Ectrodactyly and proximal/intermediate interstitial deletion 7q

    SciTech Connect

    McElveen, C.; Carvajal, M.V.; Moscatello, D.

    1995-03-13

    We report on an individual with severe mental retardation, seizures, microcephaly, unusual face, scoliosis, and cleft feet and cleft right hand. The chromosomal study showed a proximal interstitial deletion 7q (q11.23q22). From our review of the literature, 11 patients have been reported with ectrodactyly (split hand/split foot malformation) and proximal/intermediate interstitial deletions or rearrangements of 7q. The critical segment for ectrodactyly seems to be located between 7q21.2 and 7q22.1. This malformation is present in 41% of the patients whose deletion involves the critical segment. 37 refs., 3 figs., 1 tab.

  2. 22q11.2 deletion syndrome

    PubMed Central

    McDonald-McGinn, Donna M.; Sullivan, Kathleen E.; Marino, Bruno; Philip, Nicole; Swillen, Ann; Vorstman, Jacob A. S.; Zackai, Elaine H.; Emanuel, Beverly S.; Vermeesch, Joris R.; Morrow, Bernice E.; Scambler, Peter J.; Bassett, Anne S.

    2016-01-01

    22q11.2 deletion syndrome (22q11.2DS) is the most common chromosomal microdeletion disorder, estimated to result mainly from de novo non-homologous meiotic recombination events occurring in approximately 1 in every 1,000 fetuses. The first description in the English language of the constellation of findings now known to be due to this chromosomal difference was made in the 1960s in children with DiGeorge syndrome, who presented with the clinical triad of immunodeficiency, hypoparathyroidism and congenital heart disease. The syndrome is now known to have a heterogeneous presentation that includes multiple additional congenital anomalies and later-onset conditions, such as palatal, gastrointestinal and renal abnormalities, autoimmune disease, variable cognitive delays, behavioural phenotypes and psychiatric illness — all far extending the original description of DiGeorge syndrome. Management requires a multidisciplinary approach involving paediatrics, general medicine, surgery, psychiatry, psychology, interventional therapies (physical, occupational, speech, language and behavioural) and genetic counselling. Although common, lack of recognition of the condition and/or lack of familiarity with genetic testing methods, together with the wide variability of clinical presentation, delays diagnosis. Early diagnosis, preferably prenatally or neonatally, could improve outcomes, thus stressing the importance of universal screening. Equally important, 22q11.2DS has become a model for understanding rare and frequent congenital anomalies, medical conditions, psychiatric and developmental disorders, and may provide a platform to better understand these disorders while affording opportunities for translational strategies across the lifespan for both patients with 22q11.2DS and those with these associated features in the general population. PMID:27189754

  3. Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self- reactive T cells

    PubMed Central

    1996-01-01

    The Nur77/Nurr1 family of DNA binding proteins has been reported to be required for the signal transduction of CD3/T cell receptor (TCR)- mediated apoptosis in T cell hybridomas. To determine the role of this family of DNA-binding proteins in thymic clonal deletion, transgenic (Tg) mice bearing a dominant negative mutation were produced. The transgene consisted of a truncated Nur77 (deltaNur77) gene encoding the DNA-binding domain of Nur77 ligated to a TCR-beta enhancer resulting in early expression in thymocytes. Apoptosis of CD4+CD8+ thymocytes mediated by CD3/TCR signaling was greatly inhibited in the deltaNur77 Tg mice, compared with non-Tg littermates, after treatment with anti- CD3 or anti-TCR antibody in vivo and in vitro. Clonal deletion of self- reactive T cells was investigated in deltaNur77-Db/HY TCR-alpha/beta double Tg mice. There was a five-fold increase in the total number of thymocytes expressing self-reactive Db/HY TCR-alpha/beta in the deltaNur77-TCR-alpha/beta double Tg male mice. Deficient clonal deletion of self-reactive thymocytes was demonstrated by a 10-fold increase in the CD4+CD8+ thymocytes that expressed Tg TCR-alpha/beta. There was an eightfold increase in the CD8+, Db/HY TCR-alpha/beta T cells in the lymph nodes (LN) of delta Nur77-Db/HY TCR-alpha/beta double Tg compared with Db/HY TCR-alpha/beta Tg male mice. In spite of defective clonal deletion, the T cells expressing the Tg TCR were functionally anergic. In vivo analysis revealed increased activation and apoptosis of T cells associated with increased expression of Fas and Fas ligand in LN of deltaNur77-Db/HY TCR-alpha/beta double male mice. These results indicate that inhibition of Nur77/Nurr1 DNA binding in T cells leads to inefficient thymic clonal deletion, but T cell tolerance is maintained by Fas-dependent clonal deletion in LN and spleen. PMID:8666944

  4. GSTT1 Deletion Is Related to Polycyclic Aromatic Hydrocarbons-Induced DNA Damage and Lymphoma Progression

    PubMed Central

    Jia, Xiao-E; Gu, Zhao-Hui; Shi, Jing-Yi; Zhao, Yan; Li, Jun-Min; Chen, Sai-Juan; Zhao, Wei-Li

    2014-01-01

    The interrelationship between genetic susceptibility and carcinogenic exposure is important in cancer development. Polymorphisms in detoxification enzymes of the glutathione-S-transferases (GST) family are associated with an increased incidence of lymphoma. Here we investigated the molecular connection of the genetic polymorphism of GSTT1 to the response of lymphocytes to polycyclic aromatic hydrocarbons (PAH). In neoplastic situation, GSTT1 deletions were more frequently observed in lymphoma patients (54.9%) than in normal controls (42.0%, P = 0.009), resulting in an increased risk for lymphoma in individuals with GSTT1-null genotype (Odds ratio = 1.698, 95% confidence interval = 1.145–2.518). GSTT1 gene and protein expression were accordingly decreased in GSTT1-deleting patients, consistent with activated profile of cell cycle regulation genes. Mimicking environmental exposure using long-term repeat culture with low-dose PAH metabolite Hydroquinone, malignant B- and T-lymphocytes presented increased DNA damage, pCHK1/MYC expression and cell proliferation, which were counteracted by ectopic expression of GSTT1. Moreover, GSTT1 expression retarded xenograft tumor formation of Hydroquinone-treated lymphoma cells in nude mice. In non-neoplastic situation, when zebrafish was exposed to PAH Benzo(a)pyrene, molecular silencing of gstt1 enhanced the proliferation of normal lymphocytes and upregulated myca expression. Collectively, these findings suggested that GSTT1 deletion is related to genetic predisposition to lymphoma, particularly interacting with environmental pollutants containing PAH. PMID:24586676

  5. GSTT1 deletion is related to polycyclic aromatic hydrocarbons-induced DNA damage and lymphoma progression.

    PubMed

    Yang, Fan; Xiong, Jie; Jia, Xiao-E; Gu, Zhao-Hui; Shi, Jing-Yi; Zhao, Yan; Li, Jun-Min; Chen, Sai-Juan; Zhao, Wei-Li

    2014-01-01

    The interrelationship between genetic susceptibility and carcinogenic exposure is important in cancer development. Polymorphisms in detoxification enzymes of the glutathione-S-transferases (GST) family are associated with an increased incidence of lymphoma. Here we investigated the molecular connection of the genetic polymorphism of GSTT1 to the response of lymphocytes to polycyclic aromatic hydrocarbons (PAH). In neoplastic situation, GSTT1 deletions were more frequently observed in lymphoma patients (54.9%) than in normal controls (42.0%, P = 0.009), resulting in an increased risk for lymphoma in individuals with GSTT1-null genotype (Odds ratio = 1.698, 95% confidence interval = 1.145-2.518). GSTT1 gene and protein expression were accordingly decreased in GSTT1-deleting patients, consistent with activated profile of cell cycle regulation genes. Mimicking environmental exposure using long-term repeat culture with low-dose PAH metabolite Hydroquinone, malignant B- and T-lymphocytes presented increased DNA damage, pCHK1/MYC expression and cell proliferation, which were counteracted by ectopic expression of GSTT1. Moreover, GSTT1 expression retarded xenograft tumor formation of Hydroquinone-treated lymphoma cells in nude mice. In non-neoplastic situation, when zebrafish was exposed to PAH Benzo(a)pyrene, molecular silencing of gstt1 enhanced the proliferation of normal lymphocytes and upregulated myca expression. Collectively, these findings suggested that GSTT1 deletion is related to genetic predisposition to lymphoma, particularly interacting with environmental pollutants containing PAH.

  6. Nogo receptor deletion and multimodal exercise improve distinct aspects of recovery in cervical spinal cord injury.

    PubMed

    Harel, Noam Y; Song, Kang-Ho; Tang, Xin; Strittmatter, Stephen M

    2010-11-01

    We tested the ability of two plasticity-promoting approaches to enhance recovery in a mouse model of incomplete spinal cord injury (SCI). Genetically, we reduced myelin-mediated inhibition of neural plasticity through Nogo66-receptor (NgR) gene deletion. Behaviorally, we utilized a novel multimodal exercise training paradigm. Adult mice of wild-type or NgR-null genotype were subjected to partial lateral hemisection (LHx) at C3-C4 with the intent of producing anatomically and functionally mild deficits. Exercise training or control treatment proceeded for 14 weeks. Behavioral outcomes were assessed prior to tract tracing and histological analysis. Genotype and training exerted differing effects on performance; training improved performance on a test related to the training regimen (task-specific benefit), whereas genotype also improved performance on more generalized behaviors (task-non-specific benefit). There were no significant histological differences across genotype or training assignment with regard to lesion size or axonal tract staining. Thus either NgR gene deletion or exercise training benefits mice with mild cervical spinal injury. In this lesion model, the effects of NgR deletion and training were not synergistic for the tasks assessed. Further work is required to optimize the interaction between pharmacological and physical interventions for SCI.

  7. miR-155 Deletion in Female Mice Prevents Diet-Induced Obesity

    PubMed Central

    Gaudet, Andrew D.; Fonken, Laura K.; Gushchina, Liubov V.; Aubrecht, Taryn G.; Maurya, Santosh K.; Periasamy, Muthu; Nelson, Randy J.; Popovich, Phillip G.

    2016-01-01

    Obesity is a growing epidemic in developed countries. Obese individuals are susceptible to comorbidities, including cardiovascular disease and metabolic disorder. Increasing the ability of adipose tissue to expend excess energy could improve protection from obesity. One promising target is microRNA (miR)-155-5p. We demonstrate that deletion of miR-155 (-5p and -3p) in female mice prevents diet-induced obesity. Body weight gain did not differ between wild-type (WT) and miR-155 knockout (KO) mice fed control diet (CD); however, miR-155 KO mice fed high-fat diet (HFD) gained 56% less body weight and 74% less gonadal white adipose tissue (WAT) than WT mice. Enhanced WAT thermogenic potential, brown adipose tissue differentiation, and/or insulin sensitivity might underlie this obesity resistance. Indeed, miR-155 KO mice on HFD had 21% higher heat release than WT HFD mice. Compared to WT adipocytes, miR-155 KO adipocytes upregulated brown (Ucp1, Cidea, Pparg) and white (Fabp4, Pnpla2, AdipoQ, Fasn) adipogenic genes, and glucose metabolism genes (Glut4, Irs1). miR-155 deletion abrogated HFD-induced adipocyte hypertrophy and WAT inflammation. Therefore, miR-155 deletion increases adipogenic, insulin sensitivity, and energy uncoupling machinery, while limiting inflammation in WAT, which together could restrict HFD-induced fat accumulation. Our results identify miR-155 as a novel candidate target for improving obesity resistance. PMID:26953132

  8. Engineering validamycin production by tandem deletion of γ-butyrolactone receptor genes in Streptomyces hygroscopicus 5008.

    PubMed

    Tan, Gao-Yi; Peng, Yao; Lu, Chenyang; Bai, Linquan; Zhong, Jian-Jiang

    2015-03-01

    Paired homologs of γ-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (ΔshbR1) and 20% (ΔshbR3). By double deletion, the ΔshbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24 g/L (by 26%) and from 6.7 to 9.7 g/L(-1) d(-1) (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA-arpA homologs.

  9. Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury

    PubMed Central

    Xie, Cuicui; Ginet, Vanessa; Sun, Yanyan; Koike, Masato; Zhou, Kai; Li, Tao; Li, Hongfu; Li, Qian; Wang, Xiaoyang; Uchiyama, Yasuo; Truttmann, Anita C.; Kroemer, Guido; Puyal, Julien; Blomgren, Klas; Zhu, Changlian

    2016-01-01

    ABSTRACT Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy. PMID:26727396

  10. Myeloid-specific SIRT1 Deletion Aggravates Hepatic Inflammation and Steatosis in High-fat Diet-fed Mice

    PubMed Central

    Kim, Kyung Eun; Kim, Hwajin; Heo, Rok Won; Shin, Hyun Joo; Yi, Chin-ok; Lee, Dong Hoon; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Choi, Wan Sung

    2015-01-01

    Sirtuin 1 (SIRT1) is a mammalian NAD+-dependent protein deacetylase that regulates cellular metabolism and inflammatory response. The organ-specific deletion of SIRT1 induces local inflammation and insulin resistance in dietary and genetic obesity. Macrophage-mediated inflammation contributes to insulin resistance and metabolic syndrome, however, the macrophage-specific SIRT1 function in the context of obesity is largely unknown. C57/BL6 wild type (WT) or myeloid-specific SIRT1 knockout (KO) mice were fed a high-fat diet (HFD) or normal diet (ND) for 12 weeks. Metabolic parameters and markers of hepatic steatosis and inflammation in liver were compared in WT and KO mice. SIRT1 deletion enhanced HFD-induced changes on body and liver weight gain, and increased glucose and insulin resistance. In liver, SIRT1 deletion increased the acetylation, and enhanced HFD-induced nuclear translocation of nuclear factor kappa B (NF-κB), hepatic inflammation and macrophage infiltration. HFD-fed KO mice showed severe hepatic steatosis by activating lipogenic pathway through sterol regulatory element-binding protein 1 (SREBP-1), and hepatic fibrogenesis, as indicated by induction of connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), and collagen secretion. Myeloid-specific deletion of SIRT1 stimulates obesity-induced inflammation and increases the risk of hepatic fibrosis. Targeted induction of macrophage SIRT1 may be a good therapy for alleviating inflammation-associated metabolic syndrome. PMID:26330758

  11. Genetics Home Reference: 22q13.3 deletion syndrome

    MedlinePlus

    ... and symptoms include developmental delay, moderate to profound intellectual disability, decreased muscle tone (hypotonia), and absent or delayed ... the syndrome's characteristic signs (such as developmental delay, intellectual disability, and impaired speech). Additional genes in the deleted ...

  12. Report: Observed Conditions at Five Deleted Superfund Sites

    EPA Pesticide Factsheets

    Report #11-P-0433, August 3, 2011. Conditions at two of the five sites we visited in EPA Region 3, which had been remediated and deleted from the National Priorities List, may warrant additional attention from EPA.

  13. Genetics Home Reference: 17q12 deletion syndrome

    MedlinePlus

    ... spectrum disorder (which affects social interaction and communication), schizophrenia , anxiety, and bipolar disorder . Less commonly, 17q12 deletion ... Encyclopedia: Autism Spectrum Disorder Encyclopedia: Bipolar Disorder Encyclopedia: ... Topic: Developmental Disabilities Health Topic: Diabetes Health ...

  14. Genetics Home Reference: 19p13.13 deletion syndrome

    MedlinePlus

    ... Resources (1 link) National Human Genome Research Institute: Chromosome Abnormalities Educational Resources (5 links) MalaCards: chromosome 19p13.13 deletion syndrome March of Dimes: Chromosomal ...

  15. Directly repeated sequences associated with pathogenic mitochondrial DNA deletions.

    PubMed Central

    Johns, D R; Rutledge, S L; Stine, O C; Hurko, O

    1989-01-01

    We determined the nucleotide sequences of junctional regions associated with large deletions of mitochondrial DNA found in four unrelated individuals with a phenotype of chronic progressive external ophthalmoplegia. In each patient, the deletion breakpoint occurred within a directly repeated sequence of 13-18 base pairs, present in different regions of the normal mitochondrial genome-separated by 4.5-7.7 kilobases. In two patients, the deletions were identical. When all four repeated sequences are compared, a consensus sequence of 11 nucleotides emerges, similar to putative recombination signals, suggesting the involvement of a recombinational event. Partially deleted and normal mitochondrial DNAs were found in all tissues examined, but in very different proportions, indicating that these mutations originated before the primary cell layers diverged. Images PMID:2813377

  16. Genetics Home Reference: 1p36 deletion syndrome

    MedlinePlus

    ... shaped. People with 1p36 deletion syndrome may have vision or hearing problems. Some have abnormalities ... to affect between 1 in 5,000 and 1 in 10,000 newborns. However, this may be an underestimate because some ...

  17. Additions and deletions to the known cerambycidae (Coleoptera) of Bolivia

    USDA-ARS?s Scientific Manuscript database

    An additional 137 species and two tribes are added to the known cerambycid fauna of Bolivia while 12 species are deleted. Comments and statistics regarding the growth of knowledge on the Bolivian Cerambycid fauna and species endemicity are included....

  18. Genetics Home Reference: proximal 18q deletion syndrome

    MedlinePlus

    ... B, O'Donnell L, Gelfond J, Lancaster J, Fox PT, Hale DE. Consequences of chromsome18q deletions. Am ... Cody CM, Hardies LJ, Li J, Lancaster J, Fox PT, Stratton RF, Perry B, Hale DE. Recurrent ...

  19. 78 FR 17641 - Procurement List; Proposed Addition and Deletion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-22

    ...The Committee is proposing to add a service to the Procurement List that will be provided by a nonprofit agency employing persons who are blind or have other severe disabilities and, deletes a product previously furnished by such...

  20. Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

    SciTech Connect

    Hough, C.A., White, B.N., Holden, J.A.

    1995-04-01

    While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of {lambda} immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted from some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occurring during B-lymphocyte differentiation. The extent of the deleted regions was determined using probes from the various IGLV subgroups and they each covered at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage {lambda}V{lambda}135 to the distal part of the IGLV gene region. 35 refs., 4 figs.

  1. 75 FR 41451 - Procurement List; Proposed Additions and Deletion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-16

    ...The Committee is proposing to add services to the Procurement List that will be furnished by nonprofit agencies employing persons who are blind or have other severe disabilities and to delete a product previously furnished by such...

  2. Constitutional Ip36 deletion in a child with neuroblastoma

    SciTech Connect

    Biegel, J.A.; Zackai, E.H.; Scher, C.D.; Emanuel, B.S. Univ. of Pennsylvania, Philadelphia ); White, P.S.; Marshall, H.N.; Fujimori, Minoru; Brodeur, G.M. )

    1993-01-01

    The authors describe a child with dysmorphic features, as well as developmental and growth delay, who developed neuroblastoma at 5 mo of age. Cytogenetic analysis of blood lymphocytes revealed an interstitial deletion of 1p36.1 [r arrow] 1p36.2, which was apparent only with high-resolution banding. Molecular analysis with a collection of polymorphic DNA probes for 1p confirmed an interstitial deletion involving subbands of 1p36. Deletions of this region are a common finding in neuroblastoma cells from patients with advanced stages of disease. Therefore, these results (a) suggest that constitutional deletion of this region predisposed the patient to the development of neuroblastoma and (b) support the localization of a neuroblastoma tumor-suppressor locus to 1p36. 48 refs., 2 figs.

  3. Large mitochondrial DNA deletion in an infant with addison disease.

    PubMed

    Duran, Gloria P; Martinez-Aguayo, A; Poggi, H; Lagos, M; Gutierrez, D; Harris, P R

    2012-01-01

    Mitochondrial diseases are a group of disorders caused by mutations in nuclear DNA or mitochondrial DNA, usually involving multiple organ systems. Primary adrenal insufficiency due to mitochondrial disease is extremely infrequent and has been reported in association with mitochondrial DNA deletion syndromes such as Kearns-Sayre syndrome. To report a 3-year-old boy with Addison disease, congenital glaucoma, chronic pancreatitis, and mitochondrial myopathy due to large mitochondrial DNA deletion. Molecular analysis of mitochondrial DNA samples obtained from peripheral blood, oral mucosa, and muscle tissue. A novel large mitochondrial DNA deletion of 7,372bp was identified involving almost all genes on the big arch of mtDNA. This case reaffirms the association of adrenal insufficiency and mitochondrial DNA deletions and presents new evidence that glaucoma is another manifestation of mitochondrial diseases. Due to the genetic and clinical heterogeneity of mitochondrial disorders, molecular analysis is crucial to confirm diagnosis and to allow accurate genetic counseling.

  4. 75 FR 70909 - Procurement List Proposed Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-19

    ... Division II, Kansas City, MO. Barry S. Lineback, Director, Business Operations. BILLING CODE 6353-01-P ..., Division of Procurement, Washington, DC. Deletions Regulatory Flexibility Act Certification I certify that...

  5. 37 CFR 2.35 - Adding, deleting, or substituting bases.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... delete a basis at any time. (2) After publication, an applicant may add or substitute a basis in an application that is not the subject of an inter partes proceeding before the Trademark Trial and Appeal Board...

  6. Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells.

    PubMed

    Shapira-Nahor, Orit; Maayan, Shlomo; Peden, Keith W C; Rabinowitz, Ruth; Schlesinger, Michael; Alian, Akram; Panet, Amos

    2002-11-10

    Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication.

  7. Mitochondrial DNA deletions in patients with chronic suppurative otitis media.

    PubMed

    Tatar, Arzu; Tasdemir, Sener; Sahin, Ibrahim; Bozoglu, Ceyda; Erdem, Haktan Bagis; Yoruk, Ozgur; Tatar, Abdulgani

    2016-09-01

    The aim of this study was to investigate the 4977 and 7400 bp deletions of mitochondrial DNA in patients with chronic suppurative otitis media and to indicate the possible association of mitochondrial DNA deletions with chronic suppurative otitis media. Thirty-six patients with chronic suppurative otitis media were randomly selected to assess the mitochondrial DNA deletions. Tympanomastoidectomy was applied for the treatment of chronic suppurative otitis media, and the curettage materials including middle ear tissues were collected. The 4977 and 7400 bp deletion regions and two control regions of mitochondrial DNA were assessed by using the four pair primers. DNA was extracted from middle ear tissues and peripheral blood samples of the patients, and then polymerase chain reactions (PCRs) were performed. PCR products were separated in 2 % agarose gel. Seventeen of 36 patients had the heterozygote 4977 bp deletion in the middle ear tissue but not in peripheral blood. There wasn't any patient who had the 7400 bp deletion in mtDNA of their middle ear tissue or peripheral blood tissue. The patients with the 4977 bp deletion had a longer duration of chronic suppurative otitis media and a higher level of hearing loss than the others (p < 0.01). Long time chronic suppurative otitis media and the reactive oxygen species can cause the mitochondrial DNA deletions and this may be a predisposing factor to sensorineural hearing loss in chronic suppurative otitis media. An antioxidant drug as a scavenger agent may be used in long-term chronic suppurative otitis media.

  8. Evolution models with base substitutions, insertions, deletions, and selection

    NASA Astrophysics Data System (ADS)

    Saakian, D. B.

    2008-12-01

    The evolution model with parallel mutation-selection scheme is solved for the case when selection is accompanied by base substitutions, insertions, and deletions. The fitness is assumed to be either a single-peak function (i.e., having one finite discontinuity) or a smooth function of the Hamming distance from the reference sequence. The mean fitness is calculated exactly in large-genome limit. In the case of insertions and deletions the evolution characteristics depend on the choice of reference sequence.

  9. Germline CDH1 deletions in hereditary diffuse gastric cancer families

    PubMed Central

    Oliveira, Carla; Senz, Janine; Kaurah, Pardeep; Pinheiro, Hugo; Sanges, Remo; Haegert, Anne; Corso, Giovanni; Schouten, Jan; Fitzgerald, Rebecca; Vogelsang, Holger; Keller, Gisela; Dwerryhouse, Sarah; Grimmer, Donna; Chin, Suet-Feung; Yang, Han-Kwang; Jackson, Charles E.; Seruca, Raquel; Roviello, Franco; Stupka, Elia; Caldas, Carlos; Huntsman, David

    2009-01-01

    Germline CDH1 point or small frameshift mutations can be identified in 30–50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT–PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is ∼46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families. PMID:19168852

  10. Multigenerational autosomal dominant inheritance of 5p chromosomal deletions.

    PubMed

    Zhang, Bin; Willing, Marcia; Grange, Dorothy K; Shinawi, Marwan; Manwaring, Linda; Vineyard, Marisa; Kulkarni, Shashikant; Cottrell, Catherine E

    2016-03-01

    Deletion of the short arm of chromosome 5 (5p-) is associated with phenotypic features including a cat-like cry in infancy, dysmorphic facial features, microcephaly, and intellectual disability, and when encompassing a minimal critical region, may be defined as Cri-du-Chat syndrome (CdCS). Most 5p deletions are de novo in origin, and familial cases are often associated with translocation and inversion. Herein, we report three multigenerational families carrying 5p terminal deletions of different size transmitted in an autosomal dominant manner causing variable clinical findings. Terminal 5p deletions and the mode of inheritance were clinically characterized and molecularly analyzed by a combination of microarray and fluorescence in situ hybridization analyses. Shared phenotypic features documented in this cohort included neuropsychiatric findings, poor growth, and dysmorphic facial features. This study supports newly recognized effects of aberrant SEMA5A and CTNND2 dosage on severity of autistic and cognitive phenotypes. Comparative analysis of the breakpoints narrows the critical region for the cat-like cry down to an interval less than 1 Mb encompassing a candidate gene ICE1, which regulates small nuclear RNA transcription. This study also indicates that familial terminal 5p deletion is a rare presentation displaying intra- and inter-familial phenotypic variability, the latter of which may be attributed to size and gene content of the deletion. The observed intra-familial phenotypic heterogeneity suggests that additional modifying elements including genetic and environmental factors may have an impact on the clinical manifestations observed in 5p deletion carriers, and in time, further high resolution studies of 5p deletion breakpoints will continue to aid in defining genotype-phenotype correlations.

  11. A human brain tumor-derived PDGFR-alpha deletion mutant is transforming.

    PubMed

    Clarke, I D; Dirks, P B

    2003-02-06

    Aberrant receptor tyrosine kinase signaling plays an important role in the molecular pathogenesis of brain tumors. We have been studying a previously identified human glioblastoma-derived PDGFR-alpha mutant that has an in-frame deletion in the extracellular domain, causing loss of exons 8 and 9 (PDGFR-alpha(delta8,9)). In the primary tumor, this deletion mutant receptor was shown to be amplified and overexpressed. The purpose of this study was to determine the expression, activity, localization, and transformation properties of this deletion mutant. In the absence of serum, or PDGF-AA, PDGFR-alpha(delta8,9) was phosphorylated on tyrosine residues, indicating ligand-independent autoactivation. Localization by staining and cell surface biotinylation studies revealed expression of the deletion mutant predominantly in the cytoplasm, with very little present on the cell surface. To determine if PDGFR-alpha(delta8,9) was oncogenic, we transfected wild-type and mutant receptors into Rat1 cells and performed analyses of cell growth, in vitro transformation, and subcutaneous growth in the nude mouse. PDGFR-alpha(delta8,9)-expressing cells displayed enhanced cell growth and survival in low serum, and formed foci in monolayer cultures. PDGFR-alpha(delta8,9)-expressing Rat1 cells were also tumorigenic when injected subcutaneously into nude mice. Expression of PDGFR-alpha(delta8,9) was also associated with increased c-Jun phosphorylation in the absence of PDGF ligand, demonstrating also that the mutant receptor is associated with altered intracellular signaling. These data demonstrate that PDGFR-alpha(delta8,9) is transforming, and it is the first demonstration of a naturally occurring tumor-derived mutant PDGFR-alpha with oncogenic properties.

  12. Optimal In Silico Target Gene Deletion through Nonlinear Programming for Genetic Engineering

    PubMed Central

    Hong, Chung-Chien; Song, Mingzhou

    2010-01-01

    Background Optimal selection of multiple regulatory genes, known as targets, for deletion to enhance or suppress the activities of downstream genes or metabolites is an important problem in genetic engineering. Such problems become more feasible to address in silico due to the availability of more realistic dynamical system models of gene regulatory and metabolic networks. The goal of the computational problem is to search for a subset of genes to knock out so that the activity of a downstream gene or a metabolite is optimized. Methodology/Principal Findings Based on discrete dynamical system modeling of gene regulatory networks, an integer programming problem is formulated for the optimal in silico target gene deletion problem. In the first result, the integer programming problem is proved to be NP-hard and equivalent to a nonlinear programming problem. In the second result, a heuristic algorithm, called GKONP, is designed to approximate the optimal solution, involving an approach to prune insignificant terms in the objective function, and the parallel differential evolution algorithm. In the third result, the effectiveness of the GKONP algorithm is demonstrated by applying it to a discrete dynamical system model of the yeast pheromone pathways. The empirical accuracy and time efficiency are assessed in comparison to an optimal, but exhaustive search strategy. Significance Although the in silico target gene deletion problem has enormous potential applications in genetic engineering, one must overcome the computational challenge due to its NP-hardness. The presented solution, which has been demonstrated to approximate the optimal solution in a practical amount of time, is among the few that address the computational challenge. In the experiment on the yeast pheromone pathways, the identified best subset of genes for deletion showed advantage over genes that were selected empirically. Once validated in vivo, the optimal target genes are expected to achieve higher

  13. Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis.

    PubMed

    Dever, Daniel P; Adham, Zachariah O; Thompson, Bryan; Genestine, Matthieu; Cherry, Jonathan; Olschowka, John A; DiCicco-Bloom, Emanuel; Opanashuk, Lisa A

    2016-05-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis.

  14. [Observation of radiobiological characteristics in a HepG2 cell line with mitochondrial DNA deletion].

    PubMed

    Sun, Hengwen; Pan, Yi; Zeng, Zijun; Fang, Liangyi; Zhang, Hongdan; Xie, Songxi; Li, Weixiong; Xu, Jiabin

    2015-06-01

    To study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion. HepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay. HepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness. HepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

  15. Optimal in silico target gene deletion through nonlinear programming for genetic engineering.

    PubMed

    Hong, Chung-Chien; Song, Mingzhou

    2010-02-24

    Optimal selection of multiple regulatory genes, known as targets, for deletion to enhance or suppress the activities of downstream genes or metabolites is an important problem in genetic engineering. Such problems become more feasible to address in silico due to the availability of more realistic dynamical system models of gene regulatory and metabolic networks. The goal of the computational problem is to search for a subset of genes to knock out so that the activity of a downstream gene or a metabolite is optimized. Based on discrete dynamical system modeling of gene regulatory networks, an integer programming problem is formulated for the optimal in silico target gene deletion problem. In the first result, the integer programming problem is proved to be NP-hard and equivalent to a nonlinear programming problem. In the second result, a heuristic algorithm, called GKONP, is designed to approximate the optimal solution, involving an approach to prune insignificant terms in the objective function, and the parallel differential evolution algorithm. In the third result, the effectiveness of the GKONP algorithm is demonstrated by applying it to a discrete dynamical system model of the yeast pheromone pathways. The empirical accuracy and time efficiency are assessed in comparison to an optimal, but exhaustive search strategy. Although the in silico target gene deletion problem has enormous potential applications in genetic engineering, one must overcome the computational challenge due to its NP-hardness. The presented solution, which has been demonstrated to approximate the optimal solution in a practical amount of time, is among the few that address the computational challenge. In the experiment on the yeast pheromone pathways, the identified best subset of genes for deletion showed advantage over genes that were selected empirically. Once validated in vivo, the optimal target genes are expected to achieve higher genetic engineering effectiveness than a trial

  16. A small deletion in SERPINC1 causes type I antithrombin deficiency by promoting endoplasmic reticulum stress

    PubMed Central

    Cai, Lei; Zhang, Xin; Zhai, Yu; Liu, Jianren

    2016-01-01

    Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency. PMID:27708219

  17. A small deletion in SERPINC1 causes type I antithrombin deficiency by promoting endoplasmic reticulum stress.

    PubMed

    Su, Jingjing; Shu, Liang; Zhang, Zhou; Cai, Lei; Zhang, Xin; Zhai, Yu; Liu, Jianren

    2016-11-22

    Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency.

  18. Xp22. 3 deletions in isolated familial Kallmann's syndrome

    SciTech Connect

    Hardelin, J.P.; Levilliers, J.; Legouis, R.; Petit, C. ); Young, J.; Pholsena, M.; Schaison, G. ); Kirk, J.; Bouloux, P. )

    1993-04-01

    Several familial cases of Kallmann's syndrome (KS) have been reported, among which the X-chromosome-linked mode of inheritance is the most frequent. The gene responsible for the X-linked KS has been localized to the terminal part of the X-chromosome short arm (Xp22.3 region), immediately proximal to the steroid sulfatase gene responsible for X-linked ichthyosis. Large deletions of this region have been previously shown in patients affected with both X-linked ichthyosis and KS. The authors report here the search for Xp22.3 deletions in 20 unrelated males affected with isolated X-linked KS. Only 2 deletions were found using Southern blot analysis, indicating that large deletions are uncommon in patients affected with KS alone. Both deletions were shown to include the entire KAL gene responsible for X-linked KS. The patients carrying these deletions exhibit additional clinical anomalies, which are discussed: unilateral renal aplasia, unilateral absence of vas deferens, mirror movements, and sensory neural hearing loss. 47 refs., 2 figs., 1 tab.

  19. PolyA Deletions in Hereditary Nonpolyposis Colorectal Cancer

    PubMed Central

    Kim, Kyoung-Mee; Salovaara, Reijo; Mecklin, Jukka-Pekka; Järvinen, Heikki J.; Aaltonen, Lauri A.; Shibata, Darryl

    2002-01-01

    Microsatellite instability (MSI) secondary to loss of DNA mismatch repair (MMR) is present in adenomas and colorectal carcinomas from individuals with hereditary nonpolyposis colorectal cancer (HNPCC). To better characterize when MMR loss occurs during HNPCC progression, the extent of deletions in noncoding polyA sequences were compared between 6 adenomas (all ≤1.0 cm in size) and 10 cancers. Numbers of deleted bases reflect time since loss of MMR because polyA deletions are stepwise. Adenoma deletions were nearly the same (85%) as the cancers with sum total deletions at four different polyA loci of −32.7 bases in adenomas and −38.4 bases in cancers. Intervals between negative clinical examinations and tumor removal (average of 2.1 years) were known for six tumors. There were no significant differences in the extent of deletions in tumors removed under clinical surveillance (−34.8 bases) versus tumors removed without prior negative examinations (−36.5 bases). These findings illustrate that MSI is extensive in both small adenomas, and tumors which appear after negative clinical examinations, consistent with an early loss of MMR in HNPCC, even before a gatekeeper mutation. PMID:11943734

  20. miR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair

    PubMed Central

    Mandrekar-Colucci, Shweta; Hall, Jodie C.E.; Sweet, David R.; Schmitt, Philipp J.; Xu, Xinyang; Guan, Zhen; Mo, Xiaokui; Guerau-de-Arellano, Mireia

    2016-01-01

    Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers including inflammation. microRNA (miR)-155–5p is a small, noncoding RNA that negatively regulates mRNA translation. In macrophages, miR-155-5p is induced by inflammatory stimuli and elicits a response that could be toxic after SCI. miR-155 may also independently alter expression of genes that regulate axon growth in neurons. Here, we hypothesized that miR-155 deletion would simultaneously improve axon growth and reduce neuroinflammation after SCI by acting on both neurons and macrophages. New data show that miR-155 deletion attenuates inflammatory signaling in macrophages, reduces macrophage-mediated neuron toxicity, and increases macrophage-elicited axon growth by ∼40% relative to control conditions. In addition, miR-155 deletion increases spontaneous axon growth from neurons; adult miR-155 KO dorsal root ganglion (DRG) neurons extend 44% longer neurites than WT neurons. In vivo, miR-155 deletion augments conditioning lesion-induced intraneuronal expression of SPRR1A, a regeneration-associated gene; ∼50% more injured KO DRG neurons expressed SPRR1A versus WT neurons. After dorsal column SCI, miR-155 KO mouse spinal cord has reduced neuroinflammation and increased peripheral conditioning-lesion-enhanced axon regeneration beyond the epicenter. Finally, in a model of spinal contusion injury, miR-155 deletion improves locomotor function at postinjury times corresponding with the arrival and maximal appearance of activated intraspinal macrophages. In miR-155 KO mice, improved locomotor function is associated with smaller contusion lesions and decreased accumulation of inflammatory macrophages. Collectively, these data indicate that miR-155 is a novel therapeutic target capable of simultaneously overcoming neuron-intrinsic and neuron-extrinsic barriers to repair after SCI. SIGNIFICANCE STATEMENT Axon regeneration after spinal cord injury (SCI) fails

  1. Correlations between long inverted repeat (LIR) features, deletion size and distance from breakpoint in human gross gene deletions

    PubMed Central

    Aygun, Nevim

    2015-01-01

    Long inverted repeats (LIRs) have been shown to induce genomic deletions in yeast. In this study, LIRs were investigated within ±10 kb spanning each breakpoint from 109 human gross deletions, using Inverted Repeat Finder (IRF) software. LIR number was significantly higher at the breakpoint regions, than in control segments (P < 0.001). In addition, it was found that strong correlation between 5′ and 3′ LIR numbers, suggesting contribution to DNA sequence evolution (r = 0.85, P < 0.001). 138 LIR features at ±3 kb breakpoints in 89 (81%) of 109 gross deletions were evaluated. Significant correlations were found between distance from breakpoint and loop length (r = −0.18, P < 0.05) and stem length (r = −0.18, P < 0.05), suggesting DNA strands are potentially broken in locations closer to bigger LIRs. In addition, bigger loops cause larger deletions (r = 0.19, P < 0.05). Moreover, loop length (r = 0.29, P < 0.02) and identity between stem copies (r = 0.30, P < 0.05) of 3′ LIRs were more important in larger deletions. Consequently, DNA breaks may form via LIR-induced cruciform structure during replication. DNA ends may be later repaired by non-homologous end-joining (NHEJ), with following deletion. PMID:25657065

  2. Recurrent deletions of IKZF1 in pediatric acute myeloid leukemia.

    PubMed

    de Rooij, Jasmijn D E; Beuling, Eva; van den Heuvel-Eibrink, Marry M; Obulkasim, Askar; Baruchel, André; Trka, Jan; Reinhardt, Dirk; Sonneveld, Edwin; Gibson, Brenda E S; Pieters, Rob; Zimmermann, Martin; Zwaan, C Michel; Fornerod, Maarten

    2015-09-01

    IKAROS family zinc finger 1/IKZF1 is a transcription factor important in lymphoid differentiation, and a known tumor suppressor in acute lymphoid leukemia. Recent studies suggest that IKZF1 is also involved in myeloid differentiation. To investigate whether IKZF1 deletions also play a role in pediatric acute myeloid leukemia, we screened a panel of pediatric acute myeloid leukemia samples for deletions of the IKZF1 locus using multiplex ligation-dependent probe amplification and for mutations using direct sequencing. Three patients were identified with a single amino acid variant without change of IKZF1 length. No frame-shift mutations were found. Out of 11 patients with an IKZF1 deletion, 8 samples revealed a complete loss of chromosome 7, and 3 cases a focal deletion of 0.1-0.9Mb. These deletions included the complete IKZF1 gene (n=2) or exons 1-4 (n=1), all leading to a loss of IKZF1 function. Interestingly, differentially expressed genes in monosomy 7 cases (n=8) when compared to non-deleted samples (n=247) significantly correlated with gene expression changes in focal IKZF1-deleted cases (n=3). Genes with increased expression included genes involved in myeloid cell self-renewal and cell cycle, and a significant portion of GATA target genes and GATA factors. Together, these results suggest that loss of IKZF1 is recurrent in pediatric acute myeloid leukemia and might be a determinant of oncogenesis in acute myeloid leukemia with monosomy 7. Copyright© Ferrata Storti Foundation.

  3. The phenotype associated with a large deletion on MECP2

    PubMed Central

    Bebbington, Ami; Downs, Jenny; Percy, Alan; Pineda, Mercé; Zeev, Bruria Ben; Bahi-Buisson, Nadia; Leonard, Helen

    2012-01-01

    Multiplex ligation-dependent Probe Amplification (MLPA) has become available for the detection of a large deletion on the MECP2 gene allowing genetic confirmation of previously unconfirmed cases of clinical Rett syndrome. This study describes the phenotype of those with a large deletion and compares with those with other pathogenic MECP2 mutations. Individuals were ascertained from the Australian Rett Syndrome and InterRett databases with data sourced from family and clinician questionnaires, and two case studies were constructed from the longitudinal Australian data. Regression and survival analysis were used to compare severity and age of onset of symptoms in those with and without a large deletion. Data were available for 974 individuals including 51 with a large deletion and ages ranged from 1 year 4 months to 49 years (median 9 years). Those with a large deletion were more severely affected than those with other mutation types. Specifically, individuals with large deletions were less likely to have learned to walk (OR 0.42, 95% CI: 0.22–0.79, P=0.007) and to be currently walking (OR 0.53, 95% CI: 0.26–1.10, P=0.089), and were at higher odds of being in the most severe category of gross motor function (OR 1.84, 95% CI: 0.98–3.48, P=0.057) and epilepsy (OR 2.72, 95% CI: 1.38–5.37, P=0.004). They also developed epilepsy, scoliosis, hand stereotypies and abnormal breathing patterns at an earlier age. We have described the disorder profile associated with a large deletion from the largest sample to date and have found that the phenotype is severe with motor skills particularly affected. PMID:22473088

  4. A deletion variant of the alpha2b-adrenoceptor is related to emotional memory in Europeans and Africans.

    PubMed

    de Quervain, Dominique J-F; Kolassa, Iris-Tatjana; Ertl, Verena; Onyut, P Lamaro; Neuner, Frank; Elbert, Thomas; Papassotiropoulos, Andreas

    2007-09-01

    Emotionally arousing events are recalled better than neutral events. This phenomenon, which helps us to remember important and potentially vital information, depends on the activation of noradrenergic transmission in the brain. Here we show that a deletion variant of ADRA2B, the gene encoding the alpha2b-adrenergic receptor, is related to enhanced emotional memory in healthy Swiss subjects and in survivors of the Rwandan civil war who experienced highly aversive emotional situations.

  5. Myeloid-specific deletion of tumor suppressor PTEN augments neutrophil transendothelial migration during inflammation.

    PubMed

    Sarraj, Bara; Massberg, Steffen; Li, Yitang; Kasorn, Anongnard; Subramanian, Kulandayan; Loison, Fabien; Silberstein, Leslie E; von Andrian, Ulrich; Luo, Hongbo R

    2009-06-01

    Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. PIP(3) is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced PIP(3) production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-alpha intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers.

  6. 31 CFR 363.144 - May I delete a pending transaction involving a certificate of indebtedness?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 2 2010-07-01 2010-07-01 false May I delete a pending transaction... I delete a pending transaction involving a certificate of indebtedness? (a) You may delete a pending purchase of a certificate of indebtedness initiated from your TreasuryDirect ® account. (b) You may delete...

  7. 10 CFR 9.19 - Segregation of exempt information and deletion of identifying details.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...) For records required to be made available under 5 U.S.C. 552(a)(2), the NRC shall delete information that is exempt under one or more of the exemptions cited in § 9.17. The amount of information deleted... deletions are made from parts of the record by computer, the amount of information deleted will be indicated...

  8. One in Four Individuals of African-American Ancestry Harbors a 5.5kb Deletion at chromosome 11q13.1

    PubMed Central

    Zainabadi, Kayvan; Jain, Anuja V.; Donovan, Frank X.; Elashoff, David; Rao, Nagesh P.; Murty, Vundavalli V.; Chandrasekharappa, Settara C.; Srivatsan, Eri S.

    2014-01-01

    Cloning and sequencing of 5.5kb deletion at chromosome 11q13.1 from the HeLa cells, tumorigenic hybrids and two fibroblast cell lines has revealed homologous recombination between AluSx and AluY resulting in the deletion of intervening sequences. Long-range PCR of the 5.5kb sequence in 494 normal lymphocyte samples showed heterozygous deletion in 28.3% of African- American ancestry samples but only in 4.8% of Caucasian samples (p<0.0001). This observation is strengthened by the copy number variation (CNV) data of the HapMap samples which showed that this deletion occurs in 27% of YRI (Yoruba – West African) population but none in non-African populations. The HapMap analysis further identified strong linkage disequilibrium between 5 single nucleotide polymorphisms and the 5.5kb deletion in the people of African ancestry. Computational analysis of 175kb sequence surrounding the deletion site revealed enhanced flexibility, low thermodynamic stability, high repetitiveness, and stable stem-loop/hairpin secondary structures that are hallmarks of common fragile sites. PMID:24412158

  9. The BIM deletion polymorphism: A paradigm of a permissive interaction between germline and acquired TKI resistance factors in chronic myeloid leukemia.

    PubMed

    Ko, Tun Kiat; Chin, Hui San; Chuah, Charles T H; Huang, John W J; Ng, King-Pan; Khaw, Seong Lin; Huang, David C S; Ong, S Tiong

    2016-01-19

    Both germline polymorphisms and tumor-specific genetic alterations can determine the response of a cancer to a given therapy. We previously reported a germline deletion polymorphism in the BIM gene that was sufficient to mediate intrinsic resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), as well as other cancers [1]. The deletion polymorphism favored the generation of BIM splice forms lacking the pro-apoptotic BH3 domain, conferring a relative resistance to the TKI imatinib (IM). However, CML patients with the BIM deletion polymorphism developed both partial and complete IM resistance. To understand the mechanisms underlying the latter, we grew CML cells either with or without the BIM deletion polymorphism in increasing IM concentrations. Under these conditions, the BIM deletion polymorphism enhanced the emergence of populations with complete IM resistance, mimicking the situation in patients. Importantly, the combined use of TKIs with the BH3 mimetic ABT-737 overcame the BCR-ABL1-dependent and -independent resistance mechanisms found in these cells. Our results illustrate the interplay between germline and acquired genetic factors in confering TKI resistance, and suggest a therapeutic strategy for patients with complete TKI resistance associated with the BIM deletion polymorphism.

  10. Functional Genomics Using the Saccharomyces cerevisiae Yeast Deletion Collections.

    PubMed

    Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

    2016-09-01

    Constructed by a consortium of 16 laboratories, the Saccharomyces genome-wide deletion collections have, for the past decade, provided a powerful, rapid, and inexpensive approach for functional profiling of the yeast genome. Loss-of-function deletion mutants were systematically created using a polymerase chain reaction (PCR)-based gene deletion strategy to generate a start-to-stop codon replacement of each open reading frame by homologous recombination. Each strain carries two molecular barcodes that serve as unique strain identifiers, enabling their growth to be analyzed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays or through the use of next-generation sequencing technologies. Functional profiling of the deletion collections, using either strain-by-strain or parallel assays, provides an unbiased approach to systematically survey the yeast genome. The Saccharomyces yeast deletion collections have proved immensely powerful in contributing to the understanding of gene function, including functional relationships between genes and genetic pathways in response to diverse genetic and environmental perturbations. © 2016 Cold Spring Harbor Laboratory Press.

  11. Mitochondrial DNA deletion analysis: a comparison of PCR quantitative methods.

    PubMed

    Hamblet, N S; Castora, F J

    1995-02-15

    The role of mitochondrial DNA (mtDNA) deletions in aging and in neurodegenerative diseases is often determined by measuring the amount of deleted mtDNA in the affected tissue. Upon examining brain autopsy tissue from a 59 year old individual with lung cancer we determined by serial dilution PCR and kinetic PCR that a greater ratio of deleted mtDNA was present in the caudate than in the parietal cortex. However, the magnitude difference for these two brain regions appeared to be technique dependent; by serial dilution PCR the caudate had 10 times more deleted mtDNA than the parietal cortex (0.0141 vs 0.0014) whereas kinetic PCR yielded a 4-fold difference (0.1258 vs 0.0316). These results indicate that although it is valid to compare the amount of deleted mtDNA in normal and diseased tissue and draw conclusions based on relative comparisons within one study, greater caution should be exercised when comparing absolute values from studies using different measurement techniques.

  12. TransFlip Mutations Produce Deletions in Pancreatic Cancer

    PubMed Central

    Norris, Alexis L.; Kamiyama, Hirohiko; Makohon-Moore, Alvin; Pallavajjala, Aparna; Morsberger, Laura A.; Lee, Kurt; Batista, Denise; Iacobuzio-Donahue, Christine A.; Lin, Ming-Tseh; Klein, Alison P.; Hruban, Ralph H.; Wheelan, Sarah J.; Eshleman, James R.

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is driven by the inactivation of the tumor suppressor genes (TSGs), CDKN2A (P16) and SMAD4 (DPC4), commonly by homozygous deletions (HDs). Using a combination of high density single-nucleotide polymorphism (SNP) microarray and whole genome sequencing (WGS), we fine-mapped novel breakpoints surrounding deletions of CDKN2A and SMAD4 and characterized them by their underlying structural variants (SVs). Only one third of CDKN2A and SMAD4 deletions (6 of 18) were simple interstitial deletions, rather, the majority of deletions were caused by complex rearrangements, specifically, a translocation on one side of the TSG in combination with an inversion on the other side. We designate these as “TransFlip” mutations. Characteristics of TransFlip mutations are: (1) a propensity to target the TSGs CDKN2A and SMAD4 (P < 0.005), (2) not present in the germline of the examined samples, (3) non-recurrent breakpoints, (4) relatively small (47 bp to 3.4 kb) inversions, (5) inversions can be either telomeric or centromeric to the TSG, and (6) non-reciprocal, and non-recurrent translocations. TransFlip mutations are novel complex genomic rearrangements with unique breakpoint signatures in pancreatic cancer. We hypothesize that they are a common but poorly understood mechanism of TSG inactivation in human cancer. PMID:26031834

  13. The Yeast Deletion Collection: A Decade of Functional Genomics

    PubMed Central

    Giaever, Guri; Nislow, Corey

    2014-01-01

    The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MATa and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general. PMID:24939991

  14. Mouse models for the Wolf-Hirschhorn deletion syndrome.

    PubMed

    Näf, D; Wilson, L A; Bergstrom, R A; Smith, R S; Goodwin, N C; Verkerk, A; van Ommen, G J; Ackerman, S L; Frankel, W N; Schimenti, J C

    2001-01-15

    Wolf-Hirschhorn syndrome (WHS) is a deletion syndrome caused by segmental haploidy of chromosome 4p16.3. Its hallmark features include a 'Greek warrior helmet' facial appearance, mental retardation, various midline defects and seizures. The WHS critical region (WHSCR) lies between the Huntington's disease gene, HD, and FGFR3. In mice, the homologs of these genes map to chromosome 5 in a region of conserved synteny with human 4p16.3. To derive mouse models of WHS and map genes responsible for subphenotypes of the syndrome, five mouse lines bearing radiation-induced deletions spanning the WHSCR syntenic region were generated and characterized. Similar to WHS patients, these animals were growth-retarded, were susceptible to seizures and showed midline (palate closure, tail kinks), craniofacial and ocular anomalies (colobomas, corneal opacities). Other phenotypes included cerebellar hypoplasia and a shortened cerebral cortex. Expression of WHS-like traits was variable and influenced by strain background and deletion size. These mice represent the first animal models for WHS. This collection of nested chromosomal deletions will be useful for mapping and identifying loci responsible for the various subphenotypes of WHS, and provides a paradigm for the dissection of other deletion syndromes using the mouse.

  15. Molecular mimicry and clonal deletion: A fresh look.

    PubMed

    Rose, Noel R

    2015-06-21

    In this article, I trace the historic background of clonal deletion and molecular mimicry, two major pillars underlying our present understanding of autoimmunity and autoimmune disease. Clonal deletion originated as a critical element of the clonal selection theory of antibody formation in order to explain tolerance of self. If we did have complete clonal deletion, there would be major voids, the infamous "black holes", in our immune repertoire. For comprehensive, protective adaptive immunity, full deletion is necessarily a rare event. Molecular mimicry, the sharing of epitopes among self and non-self antigens, is extraordinary common and provides the evidence that complete deletion of self-reactive clones is rare. If molecular mimicry were not common, protective adaptive immunity could not be all-encompassing. By taking a fresh look at these two processes together we can envision their evolutionary basis and understand the need for regulatory devices to prevent molecular mimicry from progressing to autoimmune disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Functional Profiling Using the Saccharomyces Genome Deletion Project Collections.

    PubMed

    Nislow, Corey; Wong, Lai Hong; Lee, Amy Huei-Yi; Giaever, Guri

    2016-09-01

    The ability to measure and quantify the fitness of an entire organism requires considerably more complex approaches than simply using traditional "omic" methods that examine, for example, the abundance of RNA transcripts, proteins, or metabolites. The yeast deletion collections represent the only systematic, comprehensive set of null alleles for any organism in which such fitness measurements can be assayed. Generated by the Saccharomyces Genome Deletion Project, these collections allow the systematic and parallel analysis of gene functions using any measurable phenotype. The unique 20-bp molecular barcodes engineered into the genome of each deletion strain facilitate the massively parallel analysis of individual fitness. Here, we present functional genomic protocols for use with the yeast deletion collections. We describe how to maintain, propagate, and store the deletion collections and how to perform growth fitness assays on single and parallel screening platforms. Phenotypic fitness analyses of the yeast mutants, described in brief here, provide important insights into biological functions, mechanisms of drug action, and response to environmental stresses. It is important to bear in mind that the specific assays described in this protocol represent some of the many ways in which these collections can be assayed, and in this description particular attention is paid to maximizing throughput using growth as the phenotypic measure.

  17. The yeast deletion collection: a decade of functional genomics.

    PubMed

    Giaever, Guri; Nislow, Corey

    2014-06-01

    The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, and haploids of both MAT A: and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general. Copyright © 2014 by the Genetics Society of America.

  18. Deletion 2q37 syndrome: Cognitive-behavioral trajectories and autistic features related to breakpoint and deletion size.

    PubMed

    Fisch, Gene S; Falk, Rena E; Carey, John C; Imitola, Jaime; Sederberg, Maria; Caravalho, Karen S; South, Sarah

    2016-09-01

    Subtelomeric deletions have been reported in ∼2.5% of individuals with developmental disabilities. Subtelomeric deletion 2q37 has been detected in many individuals diagnosed with intellectual disabilities (ID) and autism spectrum disorders (ASD). Previously, genotype-phenotype correspondences were examined for their relationship to breakpoints 37.1, 37.2, or 37.3. Our purpose was to ascertain whether there were phenotypic differences at these breakpoints, elucidate the cognitive-behavioral phenotype in del2q37, and examine the genotype-phenotype association in the deletion with respect to cognitive-behavioral profiles and ASD. We administered a comprehensive cognitive-behavioral battery to nine children diagnosed with del 2q37, ages 3.9-17.75 years. ID for five tested with the Stanford-Binet (4th Edition) (SBFE) ranged from severe to mild [IQ Range: 36-59]. Adaptive behavior scores from the Vineland Adaptive Behavior Scale (VABS) were much below adequate levels (DQ Range: floor value ["19"] to 55). Autism scores from the Child Autism Rating Scale (CARS) ranged from 22 [non-autistic] to 56 [extremely autistic]; 5/8 [63%] children received scores on the autism spectrum. Participants with the largest deletions, 10.1 and 9.5 Mb, attained the highest IQ and DQ scores while those with the smallest deletions, 7.9 and 6.6 Mb, made the lowest IQ and DQ scores. No association between deletion breakpoint and phenotype were found. Assessment of the various deleted regions suggested histone deacetylase 4 gene (HDAC4) was a likely candidate gene for ASD in our sample. However, two earlier reports found no association between HDAC4 haploinsufficiency and ASD. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. GENERAL ENHANCEMENT OF MUTAGENIC POTENCY OF VARIOUS MUTAGENS DUE TO DELETED GENES IN THE ΔuvrB STRAINS TA 98 AND TA 100 OF SALMONELLA COMPARED WITH STRAINS CONTAINING ONLY A POINT MUTATION IN uvrB

    EPA Science Inventory

    The two most common strains used in Ames mutagenicity assays, TA98 and TA 100, contain a �uvrB mutation designed to enhance the mutagenicity of compounds, presumably due to the loss of the nucleotide excision repair system. We showed previously that the �uvrB mutations in these s...

  20. GENERAL ENHANCEMENT OF MUTAGENIC POTENCY OF VARIOUS MUTAGENS DUE TO DELETED GENES IN THE ΔuvrB STRAINS TA 98 AND TA 100 OF SALMONELLA COMPARED WITH STRAINS CONTAINING ONLY A POINT MUTATION IN uvrB

    EPA Science Inventory

    The two most common strains used in Ames mutagenicity assays, TA98 and TA 100, contain a �uvrB mutation designed to enhance the mutagenicity of compounds, presumably due to the loss of the nucleotide excision repair system. We showed previously that the �uvrB mutations in these s...

  1. Efficient Assembly of Ribosomes Is Inhibited by Deletion of bipA in Escherichia coli

    PubMed Central

    Choudhury, Promisree

    2015-01-01

    ABSTRACT The bacterial BipA protein belongs to the EF-G family of translational GTPases and has been postulated to be either a regulatory translation factor or a ribosome assembly factor. To distinguish between these hypotheses, we analyzed the effect of bipA deletion on three phenotypes associated with ribosome assembly factors: cold sensitivity, ribosome subunit distribution, and rRNA processing. We demonstrated that a ΔbipA strain exhibits a cold-sensitive phenotype that is similar to, and synergistic with, that of a strain with a known ribosome assembly factor, deaD. Additionally, the bipA deletion strain displayed a perturbed ribosome subunit distribution when grown at low temperature, similar to that of a deaD mutant, and again, the double mutant showed additive effects. The primary ribosomal deficiency noted was a decreased level of the 50S subunit and the appearance of a presumed pre-50S particle. Finally, deletion of bipA resulted in accumulation of pre23S rRNA, as did deletion of deaD. We further found that deletion of rluC, which encodes a pseudouridine synthase that modifies the 23S rRNA at three sites, suppressed all three phenotypes of the bipA mutant, supporting and extending previous findings. Together, these results suggest that BipA is important for the correct and efficient assembly of the 50S subunit of the ribosome at low temperature but when unmodified by RluC, the ribosomes become BipA independent for assembly. IMPORTANCE The ribosome is the complex ribonucleoprotein machine responsible for protein synthesis in all cells. Although much has been learned about the structure and function of the ribosome, we do not fully understand how it is assembled or the accessory proteins that increase efficiency of biogenesis and function. This study examined one such protein, BipA. Our results indicate that BipA either directly or indirectly enhances the formation of the 50S subunit of the ribosome, particularly at low temperature. In addition, ribosomes

  2. Efficient assembly of ribosomes is inhibited by deletion of bipA in Escherichia coli.

    PubMed

    Choudhury, Promisree; Flower, Ann M

    2015-05-01

    The bacterial BipA protein belongs to the EF-G family of translational GTPases and has been postulated to be either a regulatory translation factor or a ribosome assembly factor. To distinguish between these hypotheses, we analyzed the effect of bipA deletion on three phenotypes associated with ribosome assembly factors: cold sensitivity, ribosome subunit distribution, and rRNA processing. We demonstrated that a ΔbipA strain exhibits a cold-sensitive phenotype that is similar to, and synergistic with, that of a strain with a known ribosome assembly factor, deaD. Additionally, the bipA deletion strain displayed a perturbed ribosome subunit distribution when grown at low temperature, similar to that of a deaD mutant, and again, the double mutant showed additive effects. The primary ribosomal deficiency noted was a decreased level of the 50S subunit and the appearance of a presumed pre-50S particle. Finally, deletion of bipA resulted in accumulation of pre23S rRNA, as did deletion of deaD. We further found that deletion of rluC, which encodes a pseudouridine synthase that modifies the 23S rRNA at three sites, suppressed all three phenotypes of the bipA mutant, supporting and extending previous findings. Together, these results suggest that BipA is important for the correct and efficient assembly of the 50S subunit of the ribosome at low temperature but when unmodified by RluC, the ribosomes become BipA independent for assembly. The ribosome is the complex ribonucleoprotein machine responsible for protein synthesis in all cells. Although much has been learned about the structure and function of the ribosome, we do not fully understand how it is assembled or the accessory proteins that increase efficiency of biogenesis and function. This study examined one such protein, BipA. Our results indicate that BipA either directly or indirectly enhances the formation of the 50S subunit of the ribosome, particularly at low temperature. In addition, ribosomes contain a large

  3. Physiological activation of Akt by PHLPP1 deletion protects against pathological hypertrophy

    PubMed Central

    Moc, Courtney; Taylor, Amy E.; Chesini, Gino P.; Zambrano, Cristina M.; Barlow, Melissa S.; Zhang, Xiaoxue; Gustafsson, Åsa B.; Purcell, Nicole H.

    2015-01-01

    Aims To examine the role of physiological Akt signalling in pathological hypertrophy through analysis of PHLPP1 (PH domain leucine-rich repeat protein phosphatase) knock-out (KO) mice. Methods and results To investigate the in vivo requirement for ‘physiological’ control of Akt activation in cardiac growth, we examined the effect of deleting the Akt phosphatase, PHLPP, on the induction of cardiac hypertrophy. Basal Akt phosphorylation increased nearly two-fold in the cardiomyocytes from PHLPP1 KO mice and physiological hypertrophy induced by swimming exercise was accentuated as assessed by increased heart size and myocyte cell area. In contrast, the development of pathophysiological hypertrophy induced by pressure overload and assessed by increases in heart size, myocyte cell area, and hypertrophic gene expression was attenuated. This attenuation coincided with decreased fibrosis and cell death in the KO mice. Cast moulding revealed increased capillary density basally in the KO hearts, which was further elevated relative to wild-type mouse hearts in response to pressure overload. In vitro studies with isolated myocytes in co-culture also demonstrated that PHLPP1 deletion in cardiomyocytes can enhance endothelial tube formation. Expression of the pro-angiogenic factor VEGF was also elevated basally and accentuated in response to transverse aortic constriction in hearts from KO mice. Conclusion Our data suggest that enhancing Akt activity by inhibiting its PHLPP1-mediated dephosphorylation promotes processes associated with physiological hypertrophy that may be beneficial in attenuating the development of pathological hypertrophy. PMID:25411382

  4. Rheb1 deletion in myeloid cells aggravates OVA-induced allergic inflammation in mice

    PubMed Central

    Li, Kai; Zhang, Yue; Liang, Kang Yan; Xu, Song; Zhou, Xue Juan; Tan, Kang; Lin, Jun; Bai, Xiao Chun; Yang, Cui Lan

    2017-01-01

    The small GTPase ras homolog enriched in brain (Rheb) is a downstream target of tuberous sclerosis complex 1/2 (TSC1/2) and an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), the emerging essential modulator of M1/M2 balance in macrophages. However, the role and regulatory mechanisms of Rheb in macrophage polarization and allergic asthma are not known. In the present study, we utilized a mouse model with myeloid cell-specific deletion of the Rheb1 gene and an ovalbumin (OVA)-induced allergic asthma model to investigate the role of Rheb1 in allergic asthma and macrophage polarization. Increased activity of Rheb1 and mTORC1 was observed in myeloid cells of C57BL/6 mice with OVA-induced asthma. In an OVA-induced asthma model, Rheb1-KO mice demonstrated a more serious inflammatory response, more mucus production, enhanced airway hyper-responsiveness, and greater eosinophil numbers in bronchoalveolar lavage fluid (BALF). They also showed increased numbers of bone marrow macrophages and BALF myeloid cells, elevated M2 polarization and reduced M1 polarization of macrophages. Thus, we have established that Rheb1 is critical for the polarization of macrophages and inhibition of allergic asthma. Deletion of Rheb1 enhances M2 polarization but decreases M1 polarization in alveolar macrophages, leading to the aggravation of OVA-induced allergic asthma. PMID:28225024

  5. 11p15 ICR1 Partial Deletions Associated with IGF2/H19 DMR Hypomethylation and Silver-Russell Syndrome.

    PubMed

    Abi Habib, Walid; Brioude, Frederic; Azzi, Salah; Salem, Jennifer; Das Neves, Cristina; Personnier, Claire; Chantot-Bastaraud, Sandra; Keren, Boris; Le Bouc, Yves; Harbison, Madeleine D; Netchine, Irene

    2017-01-01

    The 11p15 region harbors the IGF2/H19 imprinted domain, implicated in fetal and postnatal growth. Silver-Russell syndrome (SRS) is characterized by fetal and postnatal growth failure, and is caused principally by hypomethylation of the 11p15 imprinting control region 1 (ICR1). However, the mechanisms leading to ICR1 hypomethylation remain unknown. Maternally inherited genetic defects affecting the ICR1 domain have been associated with ICR1 hypermethylation and Beckwith-Wiedemann syndrome (an overgrowth syndrome, the clinical and molecular mirror of SRS), and paternal deletions of IGF2 enhancers have been detected in four SRS patients. However, no paternal deletions of ICR1 have ever been associated with hypomethylation of the IGF2/H19 domain in SRS. We screened for new genetic defects within the ICR1 in a cohort of 234 SRS patients with hypomethylated IGF2/H19 domain. We report deletions close to the boundaries of ICR1 on the paternal allele in one familial and two sporadic cases of SRS with ICR1 hypomethylation. These deletions are associated with hypomethylation of the remaining CBS, and decreased IGF2 expression. These results suggest that these regions are most likely required to maintain methylation after fertilization. We estimate these anomalies to occur in about 1% of SRS cases with ICR1 hypomethylation.

  6. Altered nicotine reward-associated behavior following α4 nAChR subunit deletion in ventral midbrain.

    PubMed

    Peng, Can; Engle, Staci E; Yan, Yijin; Weera, Marcus M; Berry, Jennifer N; Arvin, Matthew C; Zhao, Guiqing; McIntosh, J Michael; Chester, Julia A; Drenan, Ryan M

    2017-01-01

    Nicotinic acetylcholine receptors containing α4 subunits (α4β2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 μg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.

  7. Inducible Deletion of Protein Kinase Map4k4 in Obese Mice Improves Insulin Sensitivity in Liver and Adipose Tissues.

    PubMed

    Danai, Laura V; Flach, Rachel J Roth; Virbasius, Joseph V; Menendez, Lorena Garcia; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Czech, Michael P

    2015-07-01

    Studies in vitro suggest that mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) attenuates insulin signaling, but confirmation in vivo is lacking since Map4k4 knockout is lethal during embryogenesis. We thus generated mice with floxed Map4k4 alleles and a tamoxifen-inducible Cre/ERT2 recombinase under the control of the ubiquitin C promoter to induce whole-body Map4k4 deletion after these animals reached maturity. Tamoxifen administration to these mice induced Map4k4 deletion in all tissues examined, causing decreased fasting blood glucose concentrations and enhanced insulin signaling to AKT in adipose tissue and liver but not in skeletal muscle. Surprisingly, however, mice generated with a conditional Map4k4 deletion in adiponectin-positive adipocytes or in albumin-positive hepatocytes displayed no detectable metabolic phenotypes. Instead, mice with Map4k4 deleted in Myf5-positive tissues, including all skeletal muscles tested, were protected from obesity-induced glucose intolerance and insulin resistance. Remarkably, these mice also showed increased insulin sensitivity in adipose tissue but not skeletal muscle, similar to the metabolic phenotypes observed in inducible whole-body knockout mice. Taken together, these results indicate that (i) Map4k4 controls a pathway in Myf5-positive cells that suppresses whole-body insulin sensitivity and (ii) Map4k4 is a potential therapeutic target for improving glucose tolerance and insulin sensitivity in type 2 diabetes.

  8. Inducible Deletion of Protein Kinase Map4k4 in Obese Mice Improves Insulin Sensitivity in Liver and Adipose Tissues

    PubMed Central

    Danai, Laura V.; Roth Flach, Rachel J.; Virbasius, Joseph V.; Garcia Menendez, Lorena; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K.

    2015-01-01

    Studies in vitro suggest that mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) attenuates insulin signaling, but confirmation in vivo is lacking since Map4k4 knockout is lethal during embryogenesis. We thus generated mice with floxed Map4k4 alleles and a tamoxifen-inducible Cre/ERT2 recombinase under the control of the ubiquitin C promoter to induce whole-body Map4k4 deletion after these animals reached maturity. Tamoxifen administration to these mice induced Map4k4 deletion in all tissues examined, causing decreased fasting blood glucose concentrations and enhanced insulin signaling to AKT in adipose tissue and liver but not in skeletal muscle. Surprisingly, however, mice generated with a conditional Map4k4 deletion in adiponectin-positive adipocytes or in albumin-positive hepatocytes displayed no detectable metabolic phenotypes. Instead, mice with Map4k4 deleted in Myf5-positive tissues, including all skeletal muscles tested, were protected from obesity-induced glucose intolerance and insulin resistance. Remarkably, these mice also showed increased insulin sensitivity in adipose tissue but not skeletal muscle, similar to the metabolic phenotypes observed in inducible whole-body knockout mice. Taken together, these results indicate that (i) Map4k4 controls a pathway in Myf5-positive cells that suppresses whole-body insulin sensitivity and (ii) Map4k4 is a potential therapeutic target for improving glucose tolerance and insulin sensitivity in type 2 diabetes. PMID:25918248

  9. [Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

    PubMed

    Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

    2015-09-01

    For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

  10. Chromosome 13q deletion with Cornelia de Lange syndrome phenotype.

    PubMed

    Ngo, C T; Alhady, M; Tan, A K; Norlasiah, I Siti; Ong, G B; Chua, C N

    2007-03-01

    A 3-year-old girl with facial dysmorphic features suggestive of Cornelia de Lange syndrome was seen in the ophthalmology unit for a right leukocoria. The leukocoria was found to be caused by a large retinoblastoma and the right eye was enucleated. Chromosomal analysis revealed partial chromosome 13q deletion involving band 14 which is associated with a high risk of retinoblastoma. This case shows that patient with chromosome 13q deletion syndrome cannot be diagnosed based on dysmorphic features only. Chromosomal analysis is warranted in all infants with facial dysmorphism suggestive of Cornelia de Lange syndrome so that those with chromosome 13q deletion can be referred early for early detection of retinoblastoma.

  11. Mitochondrial DNA deletions in Alzheimer’s brains: A review

    PubMed Central

    Phillips, Nicole R.; Simpkins, James W.; Roby, Rhonda K.

    2013-01-01

    Mitochondrial dysfunction and increased oxidative stress have been associated with normal aging and possibly implicated in the etiology of late onset Alzheimer’s disease. DNA deletions, as well as other alterations, can result from oxidative damage to nucleic acids. Many studies in the last two decades have investigated the incidence of mitochondrial DNA deletions in post-mortem brain tissues of late onset Alzheimer’s disease patients as compared to age-matched normal controls. Published studies are not entirely concordant, but their differences might shed light on the heterogeneity of Alzheimer’s disease itself. Our understanding the role that mitochondrial DNA deletions plays in disease progression may provide valuable information that could someday lead to a treatment. PMID:23850329

  12. Semilobar holoprosencephaly with 21q22 deletion: an autopsy report.

    PubMed

    Mallick, Saumyaranjan; Panda, Shasanka Shekhar; Ray, Ruma; Shukla, Rashmi; Kabra, Madhulika; Agarwal, Ramesh

    2014-03-13

    Holoprosencephaly (HPE) is the most common forebrain developmental anomaly with a prevalence of 1:16 000 live-births. Possible aetiological agents include environmental factors and genetic defects such as trisomies (13, 18) and deletions (18p, 7q, 2p and 21q). This complex malformation is due to incomplete division of the cerebral hemisphere. The phenotypes of HPE include alobar, semilobar, lobar and midline interhemispheric fusion variants. Craniofacial anomalies occur in 80% of cases. Severely affected babies die in the neonatal period. Here we report an autopsied case of semilobar HPE with pituitary and adrenal agenesis with 21q22 deletion. Additional findings are noted that would help expand the spectrum of 21q22 deletion.

  13. Specific deletion of DNA sequences between preselected bases.

    PubMed Central

    Panayotatos, N; Truong, K

    1981-01-01

    Blunt-end ligation of a "filled-in" HindIII, Sal I, Ava I or Bcl I restriction site with a DNA fragment having A, G, C, or T as the terminal 3' nucleotide regenerates the corresponding restriction site. A combination of this property with the action of BAL 31 nuclease which progressively removes base-pairs from the ends of linear DNA, can generate deletions extending to desired pre-selected nucleotides, and introduces unique restriction sites at those positions. Similarly other restriction sites can be used to select for the deletion of sequences between specific di-, tri-, tetra- and penta-nucleotides. Using this method, 10 base pairs were deleted from the end of a restriction fragment carrying the late promoter for bacteriophage T7 gene 1.1, to create a molecule with a unique restriction site at the initiation codon for translation. Images PMID:6273803

  14. Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis

    PubMed Central

    Horzempa, Joseph; Shanks, Robert M.Q.; Brown, Matthew J.; Russo, Brian C.; O’Dee, Dawn M.; Nau, Gerard J.

    2011-01-01

    We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement. PMID:19879904

  15. Deletion and deletion/insertion mutations in the juxtamembrane domain of the FLT3 gene in adult acute myeloid leukemia

    PubMed Central

    Deeb, Kristin K.; Smonskey, Matthew T.; DeFedericis, HanChun; Deeb, George; Sait, Sheila N.J.; Wetzler, Meir; Wang, Eunice S.; Starostik, Petr

    2014-01-01

    In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations. PMID:25379410

  16. Bridging the Gene-Behavior Divide through Neuroimaging Deletion Syndromes: Velocardiofacial (22q11.2 Deletion) and Williams (7q11.23 Deletion) Syndromes

    PubMed Central

    Eisenberg, Daniel Paul; Jabbi, Mbemba; Berman, Karen Faith

    2010-01-01

    Investigating the relationship between genes and the neural substrates of complex human behavior promises to provide essential insight into the pathophysiology of mental disorders. One approach to this inquiry is through neuroimaging of individuals with microdeletion syndromes that manifest in specific neuropsychiatric phenotypes. Both Velocardiofacial Syndrome (VCFS) and Williams Syndrome (WS) involve haploinsufficiency of a relatively small set of identified genes on the one hand and association with distinct, clinically-relevant behavioral and cognitive profiles on the other hand. In VCFS, there is a deletion in chromosomal region 22q11.2 and a resultant predilection toward psychosis, poor arithmetic proficiency, and low performance intelligence quotients. In WS, there is a deletion in chromosomal region 7q11.23 and a resultant predilection toward hypersociability, non-social anxiety, impaired visuospatial construction, and often intellectual impairment. Structural and functional neuroimaging studies have begun not only to map these well-defined genetic alterations to systems-level brain abnormalities, but also to identify relationships between neural phenotypes and particular genes within the critical deletion regions. Though neuroimaging of both VCFS and WS presents specific, formidable methodological challenges, including comparison subject selection and accounting for neuroanatomical and vascular anomalies in patients, and many questions remain, the literature to date on these syndromes, reviewed herein, constitutes a fruitful “bottom-up” approach to defining gene-brain relationships. PMID:20206275

  17. A deletion and a duplication in distal 22q11.2 deletion syndrome region. Clinical implications and review

    PubMed Central

    Fernández, Luis; Nevado, Julián; Santos, Fernando; Heine-Suñer, Damià; Martinez-Glez, Victor; García-Miñaur, Sixto; Palomo, Rebeca; Delicado, Alicia; Pajares, Isidora López; Palomares, María; García-Guereta, Luis; Valverde, Eva; Hawkins, Federico; Lapunzina, Pablo

    2009-01-01

    Background Individuals affected with DiGeorge and Velocardiofacial syndromes present with both phenotypic diversity and variable expressivity. The most frequent clinical features include conotruncal congenital heart defects, velopharyngeal insufficiency, hypocalcemia and a characteristic craniofacial dysmorphism. The etiology in most patients is a 3 Mb recurrent deletion in region 22q11.2. However, cases of infrequent deletions and duplications with different sizes and locations have also been reported, generally with a milder, slightly different phenotype for duplications but with no clear genotype-phenotype correlation to date. Methods We present a 7 month-old male patient with surgically corrected ASD and multiple VSDs, and dysmorphic facial features not clearly suggestive of 22q11.2 deletion syndrome, and a newborn male infant with cleft lip and palate and upslanting palpebral fissures. Karyotype, FISH, MLPA, microsatellite markers segregation studies and SNP genotyping by array-CGH were performed in both patients and parents. Results Karyotype and FISH with probe N25 were normal for both patients. MLPA analysis detected a partial de novo 1.1 Mb deletion in one patient and a novel partial familial 0.4 Mb duplication in the other. Both of these alterations were located at a distal position within the commonly deleted region in 22q11.2. These rearrangements were confirmed and accurately characterized by microsatellite marker segregation studies and SNP array genotyping. Conclusion The phenotypic diversity found for deletions and duplications supports a lack of genotype-phenotype correlation in the vicinity of the LCRC-LCRD interval of the 22q11.2 chromosomal region, whereas the high presence of duplications in normal individuals supports their role as polymorphisms. We suggest that any hypothetical correlation between the clinical phenotype and the size and location of these alterations may be masked by other genetic and/or epigenetic modifying factors. PMID

  18. A deletion and a duplication in distal 22q11.2 deletion syndrome region. Clinical implications and review.

    PubMed

    Fernández, Luis; Nevado, Julián; Santos, Fernando; Heine-Suñer, Damià; Martinez-Glez, Victor; García-Miñaur, Sixto; Palomo, Rebeca; Delicado, Alicia; Pajares, Isidora López; Palomares, María; García-Guereta, Luis; Valverde, Eva; Hawkins, Federico; Lapunzina, Pablo

    2009-06-02

    Individuals affected with DiGeorge and Velocardiofacial syndromes present with both phenotypic diversity and variable expressivity. The most frequent clinical features include conotruncal congenital heart defects, velopharyngeal insufficiency, hypocalcemia and a characteristic craniofacial dysmorphism. The etiology in most patients is a 3 Mb recurrent deletion in region 22q11.2. However, cases of infrequent deletions and duplications with different sizes and locations have also been reported, generally with a milder, slightly different phenotype for duplications but with no clear genotype-phenotype correlation to date. We present a 7 month-old male patient with surgically corrected ASD and multiple VSDs, and dysmorphic facial features not clearly suggestive of 22q11.2 deletion syndrome, and a newborn male infant with cleft lip and palate and upslanting palpebral fissures. Karyotype, FISH, MLPA, microsatellite markers segregation studies and SNP genotyping by array-CGH were performed in both patients and parents. Karyotype and FISH with probe N25 were normal for both patients. MLPA analysis detected a partial de novo 1.1 Mb deletion in one patient and a novel partial familial 0.4 Mb duplication in the other. Both of these alterations were located at a distal position within the commonly deleted region in 22q11.2. These rearrangements were confirmed and accurately characterized by microsatellite marker segregation studies and SNP array genotyping. The phenotypic diversity found for deletions and duplications supports a lack of genotype-phenotype correlation in the vicinity of the LCRC-LCRD interval of the 22q11.2 chromosomal region, whereas the high presence of duplications in normal individuals supports their role as polymorphisms. We suggest that any hypothetical correlation between the clinical phenotype and the size and location of these alterations may be masked by other genetic and/or epigenetic modifying factors.

  19. A Reduction in Age-Enhanced Gluconeogenesis Extends Lifespan

    PubMed Central

    Hachinohe, Mayumi; Yamane, Midori; Akazawa, Daiki; Ohsawa, Kazuhiro; Ohno, Mayumi; Terashita, Yuzu; Masumoto, Hiroshi

    2013-01-01

    The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan. PMID:23342062

  20. A further case of brain-lung-thyroid syndrome with deletion proximal to NKX2-1.

    PubMed

    Kharbanda, Mira; Hermanns, Pia; Jones, Jeremy; Pohlenz, Joachim; Horrocks, Iain; Donaldson, Malcolm

    2017-03-07

    Brain-lung-thyroid syndrome (OMIM #610978) is associated with mutations in the NK2 homeobox 1 (NKX2-1) gene, a transcription factor important in development. 50% of patients are affected by the full triad, comprising congenital hypothyroidism, benign hereditary chorea and infant respiratory distress syndrome. Four cases have previously been reported where a patient has features consistent with brain-lung-thyroid syndrome and a chromosome 14q13 deletion adjacent to, but not disrupting, NKX2-1. We present a patient who has a phenotype consistent with brain-lung-thyroid syndrome, featuring congenital hypothyroidism and choreoathetoid movements with gross motor delay. Thyroid ultrasound showed a small-normal gland and spontaneous resolution of hypothyroidism. Array CGH revealed a de novo 14q13.2-3 deletion adjacent to but not directly involving NKX2-1. Sequencing of NKX2-1 was normal. This report highlights a further case of chromosomal deletion adjacent to NXK2-1 in a patient with a phenotype consistent with brain-lung-thyroid syndrome, and confirms that array-CGH is a useful test in the investigation of congenital hypothyroidism. Deletion of the adjacent gene MBIP in most reported cases so far may be relevant to the pathogenesis of brain-lung-thyroid syndrome. Deletion of nearby promoter or enhancer elements acting on NKX2-1 could also be an important factor. However, further work is needed to elucidate the pathogenesis of the brain-lung-thyroid phenotype in such cases.

  1. Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

    PubMed

    Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

    2016-12-27

    The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

  2. Minimum prevalence of chromosome 22q11 deletions

    SciTech Connect

    Wilson, D.I.; Cross, I.E.; Burn, J.

    1994-09-01

    Submicroscopic deletions from within chromosome 22q11 are associated with DiGeorge (DGS), velocardiofacial (VCFS) and conotruncal anomaly syndromes and isolated congenital heart defects. In 1993 our pediatric cardiologists clinically referred all children in whom a chromosome 22q11 deletion was suspected for fluorescent in situ hybridization studies using probes from the DGS critical region. 10 affected individuals have been identified to date from the children born in 1993 in the Northern Region served exclusively by our center. A further case, the subsequent pregnancy in one of these families was affected and terminated on the basis of a major heart malformation. In the years 1988-92, for which we have complete ascertainment, there were 1009 heart defects among 191,700 births (mean 202 per annum). Thus we estimate that chromosome 22q11 deletions were the cause of at least 5% of congenital heart disease. As not all children with chromosome 22q11 deletions have a heart defect, this gives an estimated minimum prevalence of 1/4000 live births.

  3. [An updated review of 1p36 deletion (monosomy) syndrome].

    PubMed

    Bello, Sabina; Rodríguez-Moreno, Antonio

    The Monosomy 1p36 deletion syndrome is part of the group of diseases known as Rare Diseases. The objective of the present work is to review the characteristics of Monosomy 1p36 deletion syndrome. The monosomy 1p36 deletion syndrome phenotype includes: dysmorphic craniofacial features; large anterior fontanelle, unibrow, deep-set eyes, epicanthus, wide nasal root/bridge, mandible hypoplasia, abnormal location of the pinna, philtrum and pointed chin; neurological alterations: seizures and hydrocephalus (in some cases). Cerebral malformations: ventricular hypertrophy, increased subarachnoid space, morphological alterations of corpus callosum, cortical atrophy, delays in myelinisation, periventricular leukomalacia and periventricular heterotopia. These alterations produce intellectual disability and delays in motor growth, communication skills, language, social and adaptive behaviour. It is Hearing and vision impairments are also observed in subjects with this syndrome, as well as alterations of cardiac, endocrine and urinary systems and alterations at skin and skeletal level. Approximately 100 cases have been documented since 1981. This rare disease is the most common subtelomeric-micro-deletion syndrome. In situ hybridization with fluorescence (FISH) and array-comparative genomic hybridization (CGH-array) are at present the two best diagnostic techniques. There is currently no effective medical treatment for this disease. Copyright © 2016 Sociedad Chilena de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.

  4. 37 CFR 2.35 - Adding, deleting, or substituting bases.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2012-07-01 2012-07-01 false Adding, deleting, or substituting bases. 2.35 Section 2.35 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE RULES OF PRACTICE IN TRADEMARK CASES The Written Application § 2.35 Adding...

  5. 77 FR 26520 - Procurement List Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

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    Federal Register 2010, 2011, 2012, 2013, 2014

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  9. 24 CFR 990.155 - Addition and deletion of units.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... URBAN DEVELOPMENT THE PUBLIC HOUSING OPERATING FUND PROGRAM Eligibility for Operating Subsidy... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Addition and deletion of units. 990.155 Section 990.155 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN...

  10. 24 CFR 990.155 - Addition and deletion of units.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... URBAN DEVELOPMENT THE PUBLIC HOUSING OPERATING FUND PROGRAM Eligibility for Operating Subsidy... 24 Housing and Urban Development 4 2012-04-01 2012-04-01 false Addition and deletion of units. 990.155 Section 990.155 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN...

  11. 24 CFR 990.155 - Addition and deletion of units.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... URBAN DEVELOPMENT THE PUBLIC HOUSING OPERATING FUND PROGRAM Eligibility for Operating Subsidy... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Addition and deletion of units. 990.155 Section 990.155 Housing and Urban Development Regulations Relating to Housing and Urban...

  12. 24 CFR 990.155 - Addition and deletion of units.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... URBAN DEVELOPMENT THE PUBLIC HOUSING OPERATING FUND PROGRAM Eligibility for Operating Subsidy... 24 Housing and Urban Development 4 2013-04-01 2013-04-01 false Addition and deletion of units. 990.155 Section 990.155 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN...

  13. 24 CFR 990.155 - Addition and deletion of units.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... URBAN DEVELOPMENT THE PUBLIC HOUSING OPERATING FUND PROGRAM Eligibility for Operating Subsidy... 24 Housing and Urban Development 4 2014-04-01 2014-04-01 false Addition and deletion of units. 990.155 Section 990.155 Housing and Urban Development REGULATIONS RELATING TO HOUSING AND URBAN...

  14. Deletion mapping of the Aequorea victoria green fluorescent protein.

    PubMed

    Dopf, J; Horiagon, T M

    1996-01-01

    Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2-232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300-500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.

  15. 75 FR 31768 - Procurement List Proposed Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-04

    .... Service Type/Location: Grounds Maintenance, National Weather Service Forecast Office, 400 Parkway Road... severe disabilities, and to delete services previously furnished by such agencies. DATES: Comments Must... are blind or have other severe disabilities. Regulatory Flexibility Act Certification I certify that...

  16. Efficient sequential repetitive gene deletions in Neurospora crassa

    USDA-ARS?s Scientific Manuscript database

    Despite its long-standing history as a model organism, Neurospora crassa has limited tools for repetitive gene deletions utilizing recyclable self-excising marker systems. Here we describe, for the first time, the functionality of a bacterial recombination system employing ß-recombinase acting on si...

  17. 75 FR 27313 - Procurement List; Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-14

    ... products are deleted from the Procurement List: Products USB Flash Drive, Flip Style NSN: 7045-01-568-4206--1 GB, no encryption. NSN: 7045-01-568-4207--1GB, with encryption. USB Flash Drive with Password Protection NSN: 7045-01-558-4983--512MB. NSN: 7045-01-558-4984--USB Flash Drive. USB Flash Drive with...

  18. Genomic anatomy of the Tyrp1 (brown) deletion complex

    SciTech Connect

    Hunsicker, Patricia R; Johnson, Dabney K

    2006-01-01

    Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22- egabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene- oor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci (l4Rn1, l4Rn2, and l4Rn3) and the fitness mutant brown-associated fitness (baf). We have also characterized misexpression of the basonuclin homologue, Bnc2, associated with the inversion-ediated coat color mutant white-based brown (Bw). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis.

  19. 75 FR 5970 - Procurement List: Proposed Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-05

    ...The Committee is proposing to add to the Procurement List a product and a service to be furnished by nonprofit agencies employing persons who are blind or have other severe disabilities, and to delete products previously furnished by such agencies. Comments Must Be Received on or Before: March 8, 2010.

  20. On Making a Distinguished Vertex Minimum Degree by Vertex Deletion

    NASA Astrophysics Data System (ADS)

    Betzler, Nadja; Bredereck, Robert; Niedermeier, Rolf; Uhlmann, Johannes

    For directed and undirected graphs, we study the problem to make a distinguished vertex the unique minimum-(in)degree vertex through deletion of a minimum number of vertices. The corresponding NP-hard optimization problems are motivated by applications concerning control in elections and social network analysis. Continuing previous work for the directed case, we show that the problem is W[2]-hard when parameterized by the graph's feedback arc set number, whereas it becomes fixed-parameter tractable when combining the parameters "feedback vertex set number" and "number of vertices to delete". For the so far unstudied undirected case, we show that the problem is NP-hard and W[1]-hard when parameterized by the "number of vertices to delete". On the positive side, we show fixed-parameter tractability for several parameterizations measuring tree-likeness, including a vertex-linear problem kernel with respect to the parameter "feedback edge set number". On the contrary, we show a non-existence result concerning polynomial-size problem kernels for the combined parameter "vertex cover number and number of vertices to delete", implying corresponding nonexistence results when replacing vertex cover number by treewidth or feedback vertex set number.

  1. Sperm mitochondrial DNA deletion in Iranian infertiles with asthenozoospermia.

    PubMed

    Bahrehmand Namaghi, I; Vaziri, H

    2017-04-01

    Asthenozoospermia is an important cause of male infertility. The mutations in sperm mitochondrial DNA (mtDNA) result in either functionless or malfunctioning some proteins, subsequently affecting sperm motility leading to asthenozoospermia. The purpose of this study was to investigate sperm mtDNA 4,977-bp deletion in infertile men with low sperm motility/immotile spermatozoa compared to healthy subjects with high sperm motility. Semen samples of 256 asthenozoospermic infertiles and 200 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap-polymerase chain reaction (Gap-PCR) was performed. The deletion was observed in 85.93% of patients with asthenozoospermia compared with 14% in controls [OR = 37.5397, 95% confidence interval = 12.937-108.9276, p < .0001]. It is concluded that there is a strong association between sperm mtDNA 4,977-bp deletion and asthenozoospermia-induced infertility in the population examined. Large-scale mtDNA deletions in spermatozoa may induce bioenergetic disorders. Nevertheless, to validate our results broader research may be needed.

  2. Characterization of frequently deleted 6q locus in prostate cancer.

    PubMed

    Sun, Mei; Srikantan, Vasantha; Ma, Lanfeng; Li, Jia; Zhang, Wei; Petrovics, Gyorgy; Makarem, Mazen; Strovel, Jeffrey W; Horrigan, Stephen G; Augustus, Meena; Sesterhenn, Isabell A; Moul, Judd W; Chandrasekharappa, Settara; Zou, Zhiqiang; Srivastava, Shiv

    2006-11-01

    The long arm of chromosome 6 is frequently deleted in diverse human neoplasms. Our previous study showed a minimum deletion region between markers D6S1056 and D6S300 on chromosome 6q in primary prostate cancer (CaP). In this study, we further refined a 200-kb minimal region of deletion (6qTSG1) centered around D6S1013 marker. The 6qTSG1 transcripts contained complex multiple splicing variants with low or absent expression in CaP cells. None of the transcripts identified contained open reading frames that code for a protein in the NCBI database. The expression of 6qTSG transcripts revealed interesting hormonal regulation relevant to CaP biology. Expression of 6q TSG transcript was induced in LNCaP cells that were cultured in charcoal-stripped serum medium suggesting an upregulation of 6qTSG transcript by androgen ablation and cell growth inhibition/apoptosis. Induction of 6qTSG1 expression in response to androgen ablation was abrogated in androgen-independent derivatives of LNCaP cells. In summary, we have defined a candidate CaP suppressor locus on chromosome 6q16.1, and deletions of this locus are frequently associated with prostate tumorigenesis. In the light of emerging role of noncoding RNAs in cancer biology including CaP, future investigations of 6qTSG11 locus is warranted.

  3. Genomic anatomy of the Tyrp1 (brown) deletion complex

    PubMed Central

    Smyth, Ian M.; Wilming, Laurens; Lee, Angela W.; Taylor, Martin S.; Gautier, Phillipe; Barlow, Karen; Wallis, Justine; Martin, Sancha; Glithero, Rebecca; Phillimore, Ben; Pelan, Sarah; Andrew, Rob; Holt, Karen; Taylor, Ruth; McLaren, Stuart; Burton, John; Bailey, Jonathon; Sims, Sarah; Squares, Jan; Plumb, Bob; Joy, Ann; Gibson, Richard; Gilbert, James; Hart, Elizabeth; Laird, Gavin; Loveland, Jane; Mudge, Jonathan; Steward, Charlie; Swarbreck, David; Harrow, Jennifer; North, Philip; Leaves, Nicholas; Greystrong, John; Coppola, Maria; Manjunath, Shilpa; Campbell, Mark; Smith, Mark; Strachan, Gregory; Tofts, Calli; Boal, Esther; Cobley, Victoria; Hunter, Giselle; Kimberley, Christopher; Thomas, Daniel; Cave-Berry, Lee; Weston, Paul; Botcherby, Marc R. M.; White, Sharon; Edgar, Ruth; Cross, Sally H.; Irvani, Marjan; Hummerich, Holger; Simpson, Eleanor H.; Johnson, Dabney; Hunsicker, Patricia R.; Little, Peter F. R.; Hubbard, Tim; Campbell, R. Duncan; Rogers, Jane; Jackson, Ian J.

    2006-01-01

    Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22-megabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene-poor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci (l4Rn1, l4Rn2, and l4Rn3) and the fitness mutant brown-associated fitness (baf). We have also characterized misexpression of the basonuclin homologue, Bnc2, associated with the inversion-mediated coat color mutant white-based brown (Bw). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis. PMID:16505357

  4. Divergent viral superantigens delete V beta 5+ T lymphocytes.

    PubMed Central

    Gollob, K J; Palmer, E

    1992-01-01

    Several murine superantigens in association with class II major histocompatibility complex proteins have been shown to cause the deletion of T cells based on the expression of particular beta-chain variable region (V beta) gene segments. We have previously shown that mice expressing the Etc-1 superantigen, encoded by an open reading frame within the 3' long terminal repeat of the endogenous mouse mammary tumor provirus (Mtv), Mtv-9, delete T cells expressing either V beta 5 or V beta 11 gene segments. Comparison of several Mtv 3' long terminal repeat open reading frame sequences has indicated that the carboxyl terminus likely encodes the V beta specificity of these proteins. Our analysis of C57BL/6 x DBA/2 recombinant inbred strains of mouse revealed three Mtv-9-negative strains that nevertheless have a low frequency of V beta 5-expressing T cells. Here we demonstrate that a second endogenous superantigen, responsible for the deletion of V beta 5-bearing T cells, is encoded by a gene mapping to Mtv-6 on chromosome 16. Surprisingly, the carboxyl-terminal sequences of the Mtv-6 and -9 superantigens are extremely divergent, in spite of the fact that they both mediate the deletion of V beta 5+ lymphocytes. Images PMID:1317583

  5. Divergent viral superantigens delete V beta 5+ T lymphocytes.

    PubMed

    Gollob, K J; Palmer, E

    1992-06-01

    Several murine superantigens in association with class II major histocompatibility complex proteins have been shown to cause the deletion of T cells based on the expression of particular beta-chain variable region (V beta) gene segments. We have previously shown that mice expressing the Etc-1 superantigen, encoded by an open reading frame within the 3' long terminal repeat of the endogenous mouse mammary tumor provirus (Mtv), Mtv-9, delete T cells expressing either V beta 5 or V beta 11 gene segments. Comparison of several Mtv 3' long terminal repeat open reading frame sequences has indicated that the carboxyl terminus likely encodes the V beta specificity of these proteins. Our analysis of C57BL/6 x DBA/2 recombinant inbred strains of mouse revealed three Mtv-9-negative strains that nevertheless have a low frequency of V beta 5-expressing T cells. Here we demonstrate that a second endogenous superantigen, responsible for the deletion of V beta 5-bearing T cells, is encoded by a gene mapping to Mtv-6 on chromosome 16. Surprisingly, the carboxyl-terminal sequences of the Mtv-6 and -9 superantigens are extremely divergent, in spite of the fact that they both mediate the deletion of V beta 5+ lymphocytes.

  6. Commentary: The Thrill of Professionalization and the Agony of Deletes

    ERIC Educational Resources Information Center

    Waite, Susan Field; Leavell, Judy A.

    2006-01-01

    Although some teacher educators hoped that the creation and use of standards would help to professionalize teaching, the discourse of standards and accountability is now being used to erode teacher education. Many teacher educators who anticipated the thrill of professionalization through standards are now experiencing the agony of deletes,…

  7. Case-Deletion Diagnostics for Nonlinear Structural Equation Models

    ERIC Educational Resources Information Center

    Lee, Sik-Yum; Lu, Bin

    2003-01-01

    In this article, a case-deletion procedure is proposed to detect influential observations in a nonlinear structural equation model. The key idea is to develop the diagnostic measures based on the conditional expectation of the complete-data log-likelihood function in the EM algorithm. An one-step pseudo approximation is proposed to reduce the…

  8. 36 CFR 1275.58 - Deletion of restricted portions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Deletion of restricted portions. 1275.58 Section 1275.58 Parks, Forests, and Public Property NATIONAL ARCHIVES AND RECORDS ADMINISTRATION NIXON PRESIDENTIAL MATERIALS PRESERVATION AND PROTECTION OF AND ACCESS TO THE...

  9. The detection of large deletions or duplications in genomic DNA.

    PubMed

    Armour, J A L; Barton, D E; Cockburn, D J; Taylor, G R

    2002-11-01

    While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications. Copyright 2002 Wiley-Liss, Inc.

  10. Gene deletion in an Italian haemophilia B subject.

    PubMed Central

    Bernardi, F; del Senno, L; Barbieri, R; Buzzoni, D; Gambari, R; Marchetti, G; Conconi, F; Panicucci, F; Positano, M; Pitruzzello, S

    1985-01-01

    DNA from 20 Italian haemophilia B patients was analysed by the Southern blotting technique and hybridisation to a factor IX cDNA probe. A large deletion of factor IX gene was detected in one patient with antibodies to the infused factor; the EcoRI pattern of the other 19 subjects examined was normal. Images PMID:4045960

  11. 76 FR 43990 - Procurement List; Proposed Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-22

    ... Deletion from the Procurement List. SUMMARY: The Committee is proposing to add products and services to the... products and services listed below from nonprofit agencies employing persons who are blind or have other... furnish the products and services to the Government. 2. If approved, the action will result in...

  12. 77 FR 65365 - Procurement List; Proposed Addition and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-26

    ...: Goodwill Easter Seals Miami Valley, Dayton, OH. Contracting Activity: U.S. Government Accountability Office... products are proposed for deletion from the Procurement List: Products Presentation Sheets, ``SmartChart...), Seattle, WA. Contracting Activity: General Services Administration, New York, NY. Computer Accessories NSN...

  13. Multigene deletions in lung adenocarcinomas from irradiated and control mice

    SciTech Connect

    Zhang, Y.; Woloschak, G.E.

    1996-06-01

    K-ras codon 12 point mutations mRb and p53 gene deletions were examined in tissues from 120 normal lungs and lung adenocarcinomas that were Formalin-treated and paraffin-embedded 25 years ago. The results showed that 12 of 60 (20%) lung adenocarcinomas had mRb deletions. All lung adenocarcinomas that were initially found bearing deleted mRb had p53 deletions (15 of 15; 100%). A significantly higher mutation frequency for K-ras codon 12 point mutations was also found in the lung adenocarcinomas from mice exposed to 24 once-weekly neutron irradiation (10 of 10; 100%) compared with those exposed to 24 or 60 once-weekly {gamma}-ray doses (5 of 10; 50%). The data suggested that p53 and K-ras gene alterations were two contributory factors responsible for the increased incidence of lung adenocarcinoma in B6CF{sub 1} male mice exposed to protracted neutron radiation.

  14. 78 FR 25970 - Procurement List; Proposed Additions and Deletion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-03

    ... PEOPLE WHO ARE BLIND OR SEVERELY DISABLED Procurement List; Proposed Additions and Deletion AGENCY: Committee for Purchase From People Who Are Blind or Severely Disabled. ACTION: Proposed Additions to and... or Before: 6/3/2013. ADDRESSES: Committee for Purchase From People Who Are Blind or Severely Disabled...

  15. 78 FR 32631 - Procurement List; Proposed Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-31

    ... PEOPLE WHO ARE BLIND OR SEVERELY DISABLED Procurement List; Proposed Additions and Deletions AGENCY: Committee for Purchase from People Who are Blind or Severely Disabled. ACTION: Proposed Additions to and... or Before: 7/1/2013. ADDRESSES: Committee for Purchase From People Who Are Blind or Severely Disabled...

  16. Oncolytic Replication of E1b-Deleted Adenoviruses

    PubMed Central

    Cheng, Pei-Hsin; Wechman, Stephen L.; McMasters, Kelly M.; Zhou, Heshan Sam

    2015-01-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viral mRNA export, and cell cycle disruption. PMID:26561828

  17. 76 FR 9555 - Procurement List; Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-18

    ... service are deleted from the Procurement List: Products NSN: 7510-01-555-6167--Inkjet printer cartridge. NSN: 7510-01-555-6168--Inkjet printer cartridge. NSN: 7510-01-555-6169--Inkjet printer cartridge. NSN: 7510-01-555-6170--compatible with Epson Part No. T018201. Tri- color. NSN:...

  18. Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

    PubMed Central

    Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

    2013-01-01

    In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

  19. Genetics Home Reference: 22q11.2 deletion syndrome

    MedlinePlus

    ... Weissman A, Gerdes M, Pinto-Martin J, Zackai EH, McDonald-McGinn DM, Emanuel BS. Autism spectrum disorders ... PubMed Central McDonald-McGinn DM, Emanuel BS, Zackai EH. 22q11.2 Deletion Syndrome. 1999 Sep 23 [updated ...

  20. 76 FR 14942 - Procurement List; Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-18

    ... nonprofit agencies employing persons who are blind or have other severe disabilities, and deletes products...-service area; clean and sanitize food service equipment, utensil cleaning, and dishwashing; clean pots... management and operational control of the DFAC. Service Type/Location: Laundry & Dry Cleaning Service,...

  1. 76 FR 30924 - Procurement List Additions and Deletions

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-27

    ... added to the Procurement List: Products NSN: 7920-01-215-6568--Towel, Highly Absorbent, Synthetic ``Shammy'' 15x15. NSN: 7920-01-215-6569--Towel, Highly Absorbent, Synthetic ``Shammy'' 20x23. NPA... products are deleted from the Procurement List: Products Paper, Toilet Tissue NSN: 8540-01-483-8992. NPA...

  2. Seven major genomic deletions of vaccinia virus Tiantan strain are sufficient to decrease pathogenicity.

    PubMed

    Li, Yiquan; Sheng, Yuan; Chu, Yunjie; Ji, Huifan; Jiang, Shuang; Lan, Tian; Li, Min; Chen, Shuang; Fan, Yuanyuan; Li, Wenjie; Li, Xiao; Sun, Lili; Jin, Ningyi

    2016-05-01

    Attenuated strain TTVAC7, as a multi-gene-deleted vaccinia virus Tiantan strain (VTT), was constructed by knocking out parts of non-essential genes related to virulence, host range and immunomodulation of VTT, and by combining double marker screening with exogenous selectable marker knockout techniques. In this study, shuttle vector plasmids pTC-EGFP, pTA35-EGFP, pTA66-EGFP, pTE-EGFP, pTB-EGFP, pTI-EGFP and pTJ-EGFP were constructed, which contained seven pairs of recombinant arms linked to the early and late strong promoter pE/L, as well as to enhanced green fluorescent protein (EGFP) as an exogenous selectable marker. BHK cells were co-transfected/infected successively with the above plasmids and VTT or gene-deleted VTT, and homologous recombination and fluorescence plaque screening methods were used to knock out the gene fragments (TC: TC7L ∼ TK2L; TA35: TA35L; TA66: TA66R; TE: TE3L ∼ TE4L; TB: TB13R; TI: TI4L; TJ: TJ2R). The Cre/LoxP system was then applied to knock out the exogenous selectable marker, and ultimately the gene-deleted attenuated strain TTVAC7 was obtained. A series of in vivo and in vitro experiments demonstrated that not only the host range of TTVAC7 could be narrowed and its toxicity weakened significantly, but its high immunogenicity was maintained at the same time. These results support the potential of TTVAC7 to be developed as a safe viral vector or vaccine.

  3. Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

    PubMed

    Nasrallah, Gheyath K; Abdelhady, Hany; Tompkins, Nicholas P; Carson, Kaitlyn R; Garduño, Rafael A

    2014-07-01

    L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted.

  4. EZH2 expression in gliomas: Correlation with CDKN2A gene deletion/ p16 loss and MIB-1 proliferation index.

    PubMed

    Purkait, Suvendu; Sharma, Vikas; Jha, Prerana; Sharma, Mehar Chand; Suri, Vaishali; Suri, Ashish; Sharma, B S; Sarkar, Chitra

    2015-10-01

    Enhancer of zeste homolog 2 (EZH2) mediated down-regulation of CDKN2A/p16 has been observed in cell lines as well as in a few carcinomas. However, there is no study correlating EZH2 expression with CDKN2A/p16 status in gliomas. Hence, the present study was conducted to evaluate EZH2 expression in astrocytic and oligodendroglial tumors and correlate with CDKN2A/p16 status as well as MIB-1 labeling index (LI). Gliomas of all grades (n = 118) were studied using immunohistochemistry to assess EZH2, p16 and MIB-1 LI and fluorescence in situ hybrization to evaluate CDKN2A gene status. EZH2 expression and CDKN2A homozygous deletion (HD) were both significantly more frequent in high-grade gliomas (HGG). Further, strong EZH2 expression (LI ≥ 25%) was significantly more common in HGGs without CDKN2A HD (48.7%; 19/39) as compared to cases with deletion (15.8%; 3/19). Loss of p16 expression was noted in 100% and 51.3% of CDKN2A deleted and non-deleted tumors, respectively. Notably, 80% (16/20) of the CDKN2A non-deleted HGGs with p16 loss had strong EZH2 expression, in contrast to only 15.8% (3/19) in the deleted group. Loss of p16 expression significantly correlated with MIB-1 LI, irrespective of EZH2 status. Thus, this study shows that EZH2 expression correlates with tumor grade in both astrocytic and oligodendroglial tumors and hence can be used as a diagnostic marker to differentiate between low and HGGs. Further, this is the first report demonstrating an inverse correlation of strong EZH2 expression with CDKN2A HD in HGGs. Loss of p16 protein expression is mostly attributable to CDKN2A HD and correlates significantly with MIB-1 LI. Notably, our study for the first time suggests a possible epigenetic mechanism of p16 loss in CDKN2A non-deleted HGGs mediated by strong EZH2 expression. A hypothetical model for control of proliferative activity in low versus HGGs is therefore proposed. © 2015 Japanese Society of Neuropathology.

  5. Deletion affecting band 7q36 not associated with holoprosencephaly

    SciTech Connect

    Ebrahim, S.A.D.; Krivchenia, E.; Mohamed, A.N.

    1994-09-01

    Although the appearance of 7q36 aberrations have been postulated to be responsible for holoprosencephaly (HPE), the presence of a de novo 7q36 deletion in fetus without HPE has not been reported. We report the first case of a fetus with 7q36 deletion but lacking HPE. Ultrasound examination of a 25-year-old G3P1 Caucasian female showed small head circumference with microcephaly at 28 weeks. Decreased amniotic fluid volume, bilateral renal dilatation and abnormal facial features were also noted. Chromosome analysis after cordocentesis showed an abnormal female karyotype with a deletion involving the chromosome band 7q36, 46,XX,del(7)(q36). Chromosome studies on the biological parents were normal. In view of the chromosome finding and after extensive counseling, the couple elected to terminate the pregnancy. The chromosome findings were confirmed by fetal blood chromosome analysis at termination. Post-mortem examination confirmed dysmorphic features including a depressed nasal bridge and large flat ears with no lobules, but no cleft lip or palate was noted. Internal abnormalities included a bicuspid pulmonary valve and abnormally located lungs. The brain weighed 190g (249 {plus_minus} 64g expected) and had symmetric cerebral hemispheres without evidence of HPE or other gross or microscopic malformation, except focal cerebellar cortical dysplasia. In summary, our patient showed a deletion of the same chromosomal band implicated in HPE but lacked HPE. This finding indicates that 7q36 deletion may be seen in the absence of HPE and suggests that other genetic mechanisms may be responsible for HPE in this setting.

  6. Generation of Targeted Deletions in the Genome of Rhodothermus marinus▿

    PubMed Central

    Bjornsdottir, Snaedis H.; Fridjonsson, Olafur H.; Hreggvidsson, Gudmundur O.; Eggertsson, Gudmundur

    2011-01-01

    The aim of this work was to develop an approach for chromosomal engineering of the thermophile Rhodothermus marinus. A selection strategy for R. marinus had previously been developed; this strategy was based on complementing a restriction-negative trpB strain with the R. marinus trpB gene. The current work identified an additional selective marker, purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp+ selection was used during the deletion of purA from the R. marinus chromosome. The alternative Ade+ selection was in turn used while deleting the endogenous trpB gene. Since both deletions are unmarked, the purA and trpB markers may be reused. Through the double deletant SB-62 (ΔtrpB ΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency in R. marinus strain SB-62 (ΔtrpB ΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes, crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5′ and 3′ homologous sequences, respectively. The resulting Trp+ transformants were colorless rather than orange-red. The correct replacement of an internal crtBI fragment with the trpB marker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in the R. marinus chromosome can be readily replaced with linear molecules in a single step by double-crossover recombination. PMID:21705543

  7. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure.

    PubMed

    LaSarge, Candi L; Santos, Victor R; Danzer, Steve C

    2015-03-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Sites in the AAV5 capsid tolerant to deletions and tandem duplications

    PubMed Central

    Hida, Kaoru; Won, Sang Y.; Di Pasquale, Giovanni; Hanes, Justin; Chiorini, John A.; Ostermeier, Marc

    2010-01-01

    Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >105 members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands. PMID:20102698

  9. Neuron-Specific Deletion of the Nf2 Tumor Suppressor Impairs Functional Nerve Regeneration

    PubMed Central

    Schulz, Alexander; Büttner, Robert; Toledo, Andrea; Baader, Stephan L.; von Maltzahn, Julia; Irintchev, Andrey; Bauer, Reinhard; Morrison, Helen

    2016-01-01

    In contrast to axons of the central nervous system (CNS), axons of the peripheral nervous system (PNS) show better, but still incomplete and often slow regeneration following injury. The tumor suppressor protein merlin, mutated in the hereditary tumor syndrome Neurofibromatosis type 2 (NF2), has recently been shown to have RhoA regulatory functions in PNS neurons—in addition to its well-characterized, growth-inhibitory activity in Schwann cells. Here we report that the conditional knockout of merlin in PNS neurons leads to impaired functional recovery of mice following sciatic nerve crush injury, in a gene-dosage dependent manner. Gross anatomical or electrophysiological alterations of sciatic nerves could not be detected. However, correlating with attenuated RhoA activation due to merlin deletion, ultrastructural analysis of nerve samples indicated enhanced sprouting of axons with reduced caliber size and increased myelination compared to wildtype animals. We conclude that deletion of the tumor suppressor merlin in the neuronal compartment of peripheral nerves results in compromised functional regeneration after injury. This mechanism could explain the clinical observation that NF2 patients suffer from higher incidences of slowly recovering facial nerve paralysis after vestibular schwannoma surgery. PMID:27467574

  10. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure

    PubMed Central

    LaSarge, Candi L.; Santos, Victor R; Danzer, Steve C.

    2015-01-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offset the reduction in boutons per axon length. These morphological changes predicts a net increase in granule cell >> CA3 innervation. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell >> CA3 communication. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. PMID:25600212

  11. Nucleotide sequences that affect replicative and transcriptional efficiencies of Sendai virus deletion mutants.

    PubMed

    Re, G G; Kingsbury, D W

    1986-05-01

    Structural features of the genomes of virus deletion mutants (DI virions) influence their replication efficiency. Among nonsegmented negative-strand RNA viruses, substitution of the genomic 3' terminus by a complementary copy of the 5' terminus (so-called "copy-back" sequence) could enhance replication either because the new 3' end is a better promoter of RNA replication or because DI RNAs that possess this sequence are incapable of acting as templates for transcription. Here we provide evidence that both mechanisms operate in mixed infections with Sendai virus DI RNAs. RNAs incapable of transcription always outgrew RNA species that were transcribed. This was true even when the 3'-terminal sequence of the untranscribed RNA was identical to the genomic 3' terminus, as in the case of an internally deleted DI genome (RNA Ra) rendered transcriptionally inert by point mutations of bases 47 and 51 at the 5' end of the positive-strand leader RNA template. Nevertheless, Ra was outgrown by a copy-back DI RNA, indicating that the 3' genomic end of Ra is a less efficient site for replication initiation than the copy-back sequence.

  12. Conditional deletion of WT1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice

    PubMed Central

    Carmona, Rita; Cañete, Ana; Cano, Elena; Ariza, Laura; Rojas, Anabel; Muñoz-Chápuli, Ramon

    2016-01-01

    Congenital diaphragmatic hernia (CDH) is a severe birth defect. Wt1-null mouse embryos develop CDH but the mechanisms regulated by WT1 are unknown. We have generated a murine model with conditional deletion of WT1 in the lateral plate mesoderm, using the G2 enhancer of the Gata4 gene as a driver. 80% of G2-Gata4Cre;Wt1fl/fl embryos developed typical Bochdalek-type CDH. We show that the posthepatic mesenchymal plate coelomic epithelium gives rise to a mesenchyme that populates the pleuroperitoneal folds isolating the pleural cavities before the migration of the somitic myoblasts. This process fails when Wt1 is deleted from this area. Mutant embryos show Raldh2 downregulation in the lateral mesoderm, but not in the intermediate mesoderm. The mutant phenotype was partially rescued by retinoic acid treatment of the pregnant females. Replacement of intermediate by lateral mesoderm recapitulates the evolutionary origin of the diaphragm in mammals. CDH might thus be viewed as an evolutionary atavism. DOI: http://dx.doi.org/10.7554/eLife.16009.001 PMID:27642710

  13. Microscopic chromosome Xp distal deletions--a challenging issue in prenatal genetic counseling.

    PubMed

    Sukenik-Halevy, Rivka; Reches, Adi; Bar-Shira, Anat; Simchoni, Sharon; Goldstein, Myriam; Orr-Ortreger, Avi; Yaron, Yuval; Ben-Shachar, Shay

    2014-06-01

    A prenatal diagnosis of chromosome X short arm deletions may present a challenge in prenatal genetic counseling. We present clinical and molecular data of carriers of Xp distal deletions. We assessed prenatal and postnatal phenotypes of individuals from three families with large Xp distal deletions and from a fourth family with a small Xp distal deletion. The work-up included karyotyping, chromosomal microarray analysis, and assessment of the X inactivation pattern. Five out of eight women with large deletions had a short stature (<3rd percentile). Subjects from one family had developmental and emotional problems. All female carriers with small deletions had markedly short stature, whereas the men had mildly short stature. Chromosomal microarray analysis revealed 11.7-19.3 Mb deletions in three families and a small ~1 Mb deletion in the fourth. The pseudoautosomal region 1 of the X chromosome was deleted in two families with large deletions. X inactivation was skewed in all tested cases with large deletions. Xp distal deletions are mainly associated with short stature. Skewing of the abnormal X chromosome may attenuate the phenotype in cases with large deletions. We suggest that prenatal evaluation in such cases should include sonographic follow-up and assessment of the X inactivation pattern. © 2014 John Wiley & Sons, Ltd.

  14. Isolation of a putative transcriptional regulator from the region of 22q11 deleted in DiGeorge syndrome, Shprintzen syndrome and familial congenital heart disease.

    PubMed

    Halford, S; Wadey, R; Roberts, C; Daw, S C; Whiting, J A; O'Donnell, H; Dunham, I; Bentley, D; Lindsay, E; Baldini, A

    1993-12-01

    A wide spectrum of birth defects are caused by deletions of the DiGeorge syndrome critical region (DGCR) at human chromosome 22q11. Over one hundred such deletions have now been examined and a minimally deleted region of 300kb defined. Within these sequences we have identified a gene expressed during human and murine embryogenesis. The gene, named TUPLE1, and its murine homologue, encodes a protein containing repeated motifs similar to the WD40 domains found in the beta-transducin/enhancer of split (TLE) family. The TUPLE1 product has several features typical of transcriptional control proteins and in particular has homology with the yeast Tup1 transcriptional regulator. We propose that haploinsufficiency for TUPLE1 is at least partly responsible for DiGeorge syndrome and related abnormalities.

  15. Association of deletion allele of insertion/deletion polymorphism in α2B adrenoceptor gene and hypertension with or without type 2 diabetes mellitus

    PubMed Central

    Tayel, Safaa I; Khader, Heba F; El-Helbawy, Nesreen G; Ibrahim, Waleed A

    2012-01-01

    Background Vascular α2B-adrenoreceptors have the potential to increase blood pressure by mediating vasoconstriction. A nine-nucleotide deletion in the receptor enhances vasoconstriction and exacerbates hypertension. The aim of this study was to determine the association between insertion/deletion (I/D) polymorphism of the α2B-adrenoceptor and hypertension with and without diabetes. Methods The study was carried out in 35 hypertensive patients with diabetes, 35 hypertensive patients without diabetes, and 30 healthy controls. Clinical data, blood lipid profiles, and I/D polymorphism were assessed. Results Hypertensive patients were significantly older, with significantly higher systolic/diastolic blood pressures and worse plasma lipid profiles than controls. The frequency of the DD genotype was significantly higher in both hypertensive patients with (77.14%, P < 0.01) and without (71.43%, P < 0.05) diabetes versus controls (40%). Also, the D allele was significantly more common in both hypertensive patients with (84.29%, P < 0.01) and without (80%, P < 0.05) diabetes versus controls (58.33%). Hypertensive patients were more likely to have the D allele with (3.83-fold) and without (2.85-fold) diabetes. The frequencies of the DD genotype and the D allele were not significantly (P > 0.05) different between the patient groups. The DD genotype was associated with significantly lower high-density lipoprotein (P = 0.001) and significantly higher low-density lipoprotein (P = 0.017) levels versus the II and ID genotypes in the hypertensive group without diabetes. Conclusion A marked and statistically significant association between DD genotype and D allele of I/D polymorphism in the α2B-adrenoceptor gene may be a risk factor for hypertension ± diabetes. The association between the DD genotype and dyslipidemia may partially explain its role in precipitating hypertension. PMID:23776387

  16. Restoration of half the normal dystrophin sequence in a double-deletion Duchenne muscular dystrophy family

    SciTech Connect

    Hoop, R.C.; Schwartz, L.S.; Hoffman, E.P.; Russo, L.S.; Riconda, D.L.

    1994-02-01

    Two male cousins with Duchenne muscular dystrophy were found to have different maternal dystrophin gene haplotypes and different deletion mutations. One propositus showed two noncontiguous deletions-one in the 5{prime}, proximal deletional hotspot region, and the other in the 3{prime}, more distal deletional hotspot region. The second propositus showed only the 5{prime} deletion. Using multiple fluorescent exon dosage and fluorescent multiplex CA repeat linkage analyses, the authors show that the mother of each propositus carries both deletions on the same grandmaternal X chromosome. This paradox is explained by a single recombinational event between the 2 deleted regions of one of the carrier`s dystrophin genes, giving rise to a son with a partially {open_quotes}repaired{close_quotes} gene retaining only the 5{prime} deletion. 20 refs., 4 figs.

  17. Velo-cardio-facial syndrome: Frequency and textent of 22q11 deletions

    SciTech Connect

    Lindsay, E.A.; Goldberg, R.; Jurecic, V.

    1995-07-03

    Velo-cardio-facial (VCFS) or Shprintzen syndrome is associated with deletions in a region of chromosome 22q11.2 also deleted in DiGeorge anomaly and some forms of congenital heart disease. Due to the variability of phenotype, the evaluation of the incidence of deletions has been hampered by uncertainty of diagnosis. In this study, 54 patients were diagnosed with VCFS by a single group of clinicians using homogeneous clinical criteria independent of the deletion status. Cell lines of these patients were established and the deletion status evaluated for three loci within the commonly deleted region at 22q11.2 using fluorescence in situ hybridization (FISH). In 81% of the patients all three loci were hemizygous. In one patient we observed a smaller interstitial deletion than that defined by the three loci. The phenotype of this patient was not different from that observed in patients with larger deletions. 22 refs., 2 figs., 1 tab.

  18. 14 CFR 1206.202 - Deletion of segregable portions of a record.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... that is exempt, the portion that is exempt from disclosure shall be deleted and the balance of the... amount of information deleted shall be indicated on the released portion of the record, unless including...

  19. 14 CFR § 1206.202 - Deletion of segregable portions of a record.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... that is exempt, the portion that is exempt from disclosure shall be deleted and the balance of the... amount of information deleted shall be indicated on the released portion of the record, unless including...

  20. 14 CFR 1206.202 - Deletion of segregable portions of a record.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... that is exempt, the portion that is exempt from disclosure shall be deleted and the balance of the... amount of information deleted shall be indicated on the released portion of the record, unless including...

  1. Short, direct repeats at the breakpoints of deletions of the retinoblastoma gene

    SciTech Connect

    Canning, S.; Dryja, T.P. )

    1989-07-01

    The authors found deletions involving the retinoblastoma gene in 12 of 49 tumors from patients with retinoblastoma or osteosarcoma. After mapping the deletion breakpoints, they found that no two breakpoints coincided. Thus, the data do not support the conclusions of others regarding the existence of a hotspot for deletion breakpoints in this gene. In 4 of the tumors, they sequenced 200 base pairs surrounding each deletion breakpoint. Three deletions had termini within pairs of short, direct repeats ranging in size from 4 to 7 base pairs. These results indicate that the slipped mispairing mechanism may predominate in the generation of deletions at this locus. The review of deletion breakpoints at other genetic loci reveals that the nature of the sequences present at deletion breakpoints (short, direct repeats versus middle repetitive elements) varies according to the genetic locus under study.

  2. Dystrophin expression in a Duchenne muscular dystrophy patient with a frame shift deletion.

    PubMed

    Prior, T W; Bartolo, C; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Kissel, J T; Luquette, M H; Tsao, C Y; Mendell, J R

    1997-02-01

    The exon 45 deletion is a common dystrophin gene deletion. Although this is an out-of-frame deletion, which should not allow for protein synthesis, it has been observed in mildly affected patients. We describe a patient with an exon 45 deletion who produced protein, but still had a severe Duchenne muscular dystrophy phenotype. RT-PCR analysis and cDNA sequencing from the muscle biopsy sample revealed that the exon 45 deletion induced exon skipping of exon 44, which resulted in an in-frame deletion and the production of dystrophin. A conformational change in dystrophin induced by the deletion is proposed as being responsible for the severe phenotype in the patient. We feel that the variable clinical phenotype observed in patients with the exon 45 deletion is not due to exon splicing but may be the result of other environmental or genetic factors, or both.

  3. Wheat streak mosaic virus Coat Protein Deletion Mutants Elicit More Severe Symptoms than Wild-Type Virus in Multiple Cereal Hosts.

    PubMed

    Tatineni, Satyanarayana; ELowsky, Christian; Graybosch, Robert

    2017-08-25

    Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84, but not 36 to 57, induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared to mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from WSMV genome accelerated cell-to-cell movement with increased accumulation of genomic RNAs and CP compared to wild-type virus. Microscopic examination of wheat tissues infected by GFP-tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis compared to mutants eliciting wild-type symptoms. All deletion mutants accumulated CP-specific major protein similar to wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared to wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from WSMV genome enhanced accumulation of CP and genomic RNA, altered CP-specific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts.

  4. Sequence Homology at the Breakpoint and Clinical Phenotype of Mitochondrial DNA Deletion Syndromes

    PubMed Central

    Sadikovic, Bekim; Wang, Jing; El-Hattab, Ayman; Landsverk, Megan; Douglas, Ganka; Brundage, Ellen K.; Craigen, William J.; Schmitt, Eric S.; Wong, Lee-Jun C.

    2010-01-01

    Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (<6 years old) showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (<6 years old) carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and

  5. A deletion hampering appropriate typing of Mycobacterium africanum.

    PubMed

    Abascal, Estefanía; Herrera, Diana Maricela; Herranz, Marta; Santantón, Sheila; Martínez-Lirola, Miguel; Tudó, Griselda; Gonzalez, Juliá; Bouza, Emilio; Pérez-Lago, Laura; García-de-Viedma, Darío

    2017-03-01

    Molecular epidemiology analysis of tuberculosis transmission is based mostly on the application of MIRU-VNTR. In certain isolates a complete 24-loci genotype is not obtained and these incompletely genotyped isolates can not be used in the definition of clusters. In a population-based molecular epidemiology study performed in Almería, Southeast Spain, a context with a high proportion of immigrants, we found that an 88-bp deletion in isolates of Mycobacterium africanum Lineage 5 hampers MIRU-VNTR analysis. A more extensive analysis revealed that this deletion was shared by all the Lineage 5 isolates in certain countries of origin of immigrants, such as Equatorial Guinea, and is likely present in several other African countries and also in the USA. A procedure is proposed to enable epidemiological analysis of these isolates.

  6. Effects of deleting A19 tyrosine from insulin.

    PubMed

    Du, X; Tang, J G

    1998-03-01

    A mutant proinsulin gene was constructed through PCR mediated mutagenesis. The code of A19Tyr was deleted. The mutant proinsulin, (deltaY)19-lys-proinsulin [(deltaY)19KPI], was expressed in E. coli and purified. After treatment with trypsin and carboxypeptidase B, and Resource Q separation, (deltaY)19-human insulin [(deltaY)19HI] was obtained. It retains 63.6% of receptor binding activity but only 2.2% of immune activity, and shows a longer retaining time on reverse-phase FPLC and a slower mobility by native PAGE analysis. These results suggest that the deletion of A19Tyr causes some conformational changes on insulin, which plays a minor role on the affinity of insulin to its receptor, and a major role on immunogenicity of the hormone.

  7. Bayesian Case-deletion Model Complexity and Information Criterion

    PubMed Central

    Zhu, Hongtu; Ibrahim, Joseph G.; Chen, Qingxia

    2015-01-01

    We establish a connection between Bayesian case influence measures for assessing the influence of individual observations and Bayesian predictive methods for evaluating the predictive performance of a model and comparing different models fitted to the same dataset. Based on such a connection, we formally propose a new set of Bayesian case-deletion model complexity (BCMC) measures for quantifying the effective number of parameters in a given statistical model. Its properties in linear models are explored. Adding some functions of BCMC to a conditional deviance function leads to a Bayesian case-deletion information criterion (BCIC) for comparing models. We systematically investigate some properties of BCIC and its connection with other information criteria, such as the Deviance Information Criterion (DIC). We illustrate the proposed methodology on linear mixed models with simulations and a real data example. PMID:26180578

  8. 5p Deletions: Current Knowledge and Future Directions

    PubMed Central

    Nguyen, Joanne M.; Qualmann, Krista J.; Okashah, Rebecca; Reilly, Amysue; Alexeyev, Mikhail F.; Campbell, Dennis J.

    2016-01-01

    Disorders resulting from 5p deletions (5p–) were first recognized by Lejeune et al. in 1963 [Lejeune et al. (1963); C R Hebd Seances Acad Sci 257:3098-3102]. 5p– is caused by partial or total deletion of the short arm of chromosome 5. The most recognizable phenotype is characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. This report reviews 5p– disorders and their molecular basis. Hemizygosity for genes located within this region have been implicated in contributing to the phenotype. A review of the genes on 5p which may be dosage sensitive is summarized. Because of the growing knowledge of these specific genes, future directions to explore potential targeted therapies for individuals with 5p– are discussed. PMID:26235846

  9. Efficient, complete deletion of synaptic proteins using CRISPR.

    PubMed

    Incontro, Salvatore; Asensio, Cedric S; Edwards, Robert H; Nicoll, Roger A

    2014-09-03

    One of the most powerful ways to test the function of a protein is to characterize the consequences of its deletion. In the past, this has involved inactivation of the gene by homologous recombination either in the germline or later through conditional deletion. RNA interference (RNAi) provides an alternative way to knock down proteins, but both of these approaches have their limitations. Recently, the CRISPR/Cas9 system has suggested another way to selectively inactivate genes. We have now tested this system in postmitotic neurons by targeting two well-characterized synaptic proteins, the obligatory GluN1 subunit of the NMDA receptor and the GluA2 subunit of the AMPA receptor. Expression of CRISPR/Cas9 in hippocampal slice cultures completely eliminated NMDA receptor and GluA2 function. CRISPR/Cas9 thus provides a powerful tool to study the function of synaptic proteins.

  10. A novel partial sequence alignment tool for finding large deletions.

    PubMed

    Aruk, Taner; Ustek, Duran; Kursun, Olcay

    2012-01-01

    Finding large deletions in genome sequences has become increasingly more useful in bioinformatics, such as in clinical research and diagnosis. Although there are a number of publically available next generation sequencing mapping and sequence alignment programs, these software packages do not correctly align fragments containing deletions larger than one kb. We present a fast alignment software package, BinaryPartialAlign, that can be used by wet lab scientists to find long structural variations in their experiments. For BinaryPartialAlign, we make use of the Smith-Waterman (SW) algorithm with a binary-search-based approach for alignment with large gaps that we called partial alignment. BinaryPartialAlign implementation is compared with other straight-forward applications of SW. Simulation results on mtDNA fragments demonstrate the effectiveness (runtime and accuracy) of the proposed method.

  11. Bayesian Case-deletion Model Complexity and Information Criterion.

    PubMed

    Zhu, Hongtu; Ibrahim, Joseph G; Chen, Qingxia

    2014-10-01

    We establish a connection between Bayesian case influence measures for assessing the influence of individual observations and Bayesian predictive methods for evaluating the predictive performance of a model and comparing different models fitted to the same dataset. Based on such a connection, we formally propose a new set of Bayesian case-deletion model complexity (BCMC) measures for quantifying the effective number of parameters in a given statistical model. Its properties in linear models are explored. Adding some functions of BCMC to a conditional deviance function leads to a Bayesian case-deletion information criterion (BCIC) for comparing models. We systematically investigate some properties of BCIC and its connection with other information criteria, such as the Deviance Information Criterion (DIC). We illustrate the proposed methodology on linear mixed models with simulations and a real data example.

  12. A case of 18p deletion syndrome after blepharoplasty

    PubMed Central

    Xu, Li-juan; Wu, Lv-xian; Yuan, Qing; Lv, Zhi-gang; Jiang, Xue-yan

    2017-01-01

    Objective The deletion of the short arm of chromosome 18 is thought to be one of the rare chromosomal aberrations. Here, we report a case to review this disease. Case report The proband is a five-and-a-half-year-old girl who has had phenotypes manifested mainly by ptosis, broad face, broad neck with low posterior hairline, mental retardation, short stature, and other malformations. Chromosomal analysis for her mother showed a normal karyotype. Her father and younger brother were phenotypically normal. Result Phenotypical features were quite similar throughout other cases and in accordance with the usual phenotype of del(18p) suggested within the same cases and among the del(18p) cases described. She underwent blepharoplasty, which improved her appearance. Conclusion 18p deletion syndrome is diagnosed by gene analysis. Plastic surgeries for improving the appearance might be an option for these patients. PMID:28138267

  13. Deleted in Breast Cancer 1 regulates cellular senescence during obesity.

    PubMed

    Escande, Carlos; Nin, Veronica; Pirtskhalava, Tamar; Chini, Claudia C; Thereza Barbosa, Maria; Mathison, Angela; Urrutia, Raul; Tchkonia, Tamar; Kirkland, James L; Chini, Eduardo N

    2014-10-01

    Chronic obesity leads to inflammation, tissue dysfunction, and cellular senescence. It was proposed that cellular senescence during obesity and aging drives inflammation and dysfunction. Consistent with this, clearance of senescent cells increases healthspan in progeroid mice. Here, we show that the protein Deleted in Breast Cancer-1 (DBC1) regulates cellular senescence during obesity. Deletion of DBC1 protects preadipocytes against cellular senescence and senescence-driven inflammation. Furthermore, we show protection against cellular senescence in DBC1 KO mice during obesity. Finally, we found that DBC1 participates in the onset of cellular senescence in response to cell damage by mechanism that involves binding and inhibition of HDAC3. We propose that by regulating HDAC3 activity during cellular damage, DBC1 participates in the fate decision that leads to the establishment of cellular senescence and consequently to inflammation and tissue dysfunction during obesity. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  14. A Family Harboring CMT1A Duplication and HNPP Deletion.

    PubMed

    Lee, Jung Hwa; Kang, Hee Jin; Song, Hyunseok; Hwang, Su Jin; Cho, Sun-Young; Kim, Sang-Beom; Kim, Joonki; Chung, Ki Wha; Choi, Byung-Ok

    2007-06-01

    Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with duplication of chromosome 17p11.2-p12, whereas hereditary neuropathy with liability to pressure palsies (HNPP), which is an autosomal dominant neuropathy showing characteristics of recurrent pressure palsies, is associated with 17p11.2-p12 deletion. An altered gene dosage of PMP22 is believed to the main cause underlying the CMT1A and HNPP phenotypes. Although CMT1A and HNPP are associated with the same locus, there has been no report of these two mutations within a single family. We report a rare family harboring CMT1A duplication and HNPP deletion.

  15. Exploration of geosmin synthase from Streptomyces peucetius ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster.

    PubMed

    Singh, Bijay; Oh, Tae-Jin; Sohng, Jae Kyung

    2009-10-01

    Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 +/- 0.4-fold enhanced production of geosmin was observed.

  16. Athymic (nude) mice fail to delete functional self-reactive helper T cells.

    PubMed

    Caulfield, M J; Stanko, D; Isaak, D D

    1993-09-01

    We have examined the thymic requirement for the antibody response to a foreign antigen coupled to self erythrocytes. We find that self erythrocytes mediate thymus-independent, carrier specific help for the antibody response to the pneumococcal cell wall polysaccharide antigen, PnC. Thus, athymic nude mice gave a high primary antibody plaque-forming cell (PFC) response to PnC-mouse RBC but a low response to PnC coupled to sheep or burro RBC. The meager response to PnC coupled to foreign RBCs could not be attributed to antigenic competition since the response to the carrier (burro RBC) was < 100 PFC per spleen. Reconstitution of nude mice with splenic T cells from euthymic mice enhanced rather than suppressed the antibody response to PnC-mouse RBC. The results document that in the absence of thymic deletion, functional self-reactive helper cells persist in the nude mouse.

  17. 76 FR 27999 - Procurement List; Addition and Deletion

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-13

    ... business the opportunity to compete for these projects in the future. The Javits-Wagner-O'Day Act and its... the objectives of the Javits-Wagner-O'Day Act (41 U.S.C. 46- 48c) in connection with the service...-Wagner-O'Day Act (41 U.S.C. 46- 48c) in connection with the service deleted from the Procurement List...

  18. Design and Implementation of a Multimedia DBMS: Modification and Deletion

    DTIC Science & Technology

    1991-09-01

    process data are discussed. This thesis concentrates on the design and implementation operations for deletion and modification of formatted and...of this thesis , only the create, insert, and retrieve options were available. The system, as it is now, performs media data processing by means of...AD-A246 080 NAVAL POSTGRADUATE SCHOOL Monterey, California DTIC ELECTE FEB 20 199211 .S D THESIS DESIGN AND IMPLEMENTATION OF A MULTIMEDIA DBMS

  19. Mitochondrial DNA deletions and depletion within paraspinal muscles

    PubMed Central

    Campbell, G R; Reeve, A; Ziabreva, I; Polvikoski, T M; Taylor, R W; Reynolds, R; Turnbull, D M; Mahad, D J

    2013-01-01

    Aims Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age-related histopathological changes were previously reported. Methods Cervical and lumbar paraspinal muscles were obtained peri-operatively from 13 patients and from six post mortem control cases (age range 18–82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory-deficient muscle fibres (lacking complex IV with intact complex II activity). Real-time polymerase chain reaction, long-range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry. Results The density of respiratory-deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory-deficient (range 0–10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory-deficient fibres with and without mtDNA deletions or mtDNA depletion. Conclusions Paraspinal muscles appeared to be particularly susceptible to age-related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age-related postural abnormalities

  20. Large deletion in the NF1 gene associated with dysmorphism

    SciTech Connect

    Hughes, H.E.; Maynard, J.; Sourour, E.

    1994-09-01

    Neurofibromatosis type 1 is an autosomal dominant disorder with a prevalence of 1 in 3000. The major clinical features of the disease include cafe-au-lait spots, neurofibromas, Lisch nodules and auxillary freckling. Six sporadic NF1 patients with dysmorphism and intellectual impairment have been described to have a large deletion extending beyond the NF1 gene. We report another spordiac NF1 patient with severe developmental delay, early growth failure and dysmorphism (not Noonan-like) associated with a large deletion involving the NF1 gene. A panel of 12 polymorphic DNA markers within 4 cM of the NF1 gene were used to screen for the NF1 gene rearrangements. With all the polymorphic markers, only a single band was ever observed in this affected individual. However, with DNA probe EW301 which maps to 17p, a biparental inheritance was observed. Analysis with several microsatellite markers indicated that this patient had not inherited an allele from the father. A reduction in the hybridization signal was also observed when DNA from this patient was screened with cDNAs AE25, P5, B3A, and an extragenic marker EW206, clearly indicating hemizygosity at these loci. The combined evidence of dosage reduction and biparental inheritance with DNA marker EW301 indictates that this patient has a deletion of paternal origin rather than uniparental disomy. Pulsed-field gel electrophoresis has not, so far, revealed any evidence of an altered band pattern; however, studies are continuing. FISH analysis is currently in progress using YACs and cosmids to define the extent of this deletion.

  1. Deletion (11)(q14.1q21)

    SciTech Connect

    Stratton, R.F.; Lazarus, K.H.; Ritchie, E.J.L.; Bell, A.M.

    1994-02-01

    The authors report on a 4-year-old girl with moderate development delay, horseshoe kidney, bilateral duplication of the ureters with right upper pole obstruction, hydronephrosis and nonfunction, and subsequent Wilms tumor of the right lower pole. She had an interstitial deletion of the long arm of chromosome 11 involving the region 11(q14.1q21). 22 refs., 2 figs., 1 tab.

  2. 47 CFR 1.229 - Motions to enlarge, change, or delete issues.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 1 2012-10-01 2012-10-01 false Motions to enlarge, change, or delete issues. 1... Hearing Proceedings Participants and Issues § 1.229 Motions to enlarge, change, or delete issues. (a) A motion to enlarge, change or delete the issues may be filed by any party to a hearing. Except as provided...

  3. Characterization of Deletions of the HBA and HBB Loci by Array Comparative Genomic Hybridization.

    PubMed

    Sabath, Daniel E; Bender, Michael A; Sankaran, Vijay G; Vamos, Esther; Kentsis, Alex; Yi, Hye-Son; Greisman, Harvey A

    2016-01-01

    Thalassemia is among the most common genetic diseases worldwide. α-Thalassemia is usually caused by deletion of one or more of the duplicated HBA genes on chromosome 16. In contrast, most β-thalassemia results from point mutations that decrease or eliminate expression of the HBB gene on chromosome 11. Deletions within the HBB locus result in thalassemia or hereditary persistence of fetal Hb. Although routine diagnostic testing cannot distinguish thalassemia deletions from point mutations, deletional hereditary persistence of fetal Hb is notable for having an elevated HbF level with a normal mean corpuscular volume. A small number of deletions accounts for most α-thalassemias; in contrast, there are no predominant HBB deletions causing β-thalassemia. To facilitate the identification and characterization of deletions of the HBA and HBB globin loci, we performed array-based comparative genomic hybridization using a custom oligonucleotide microarray. We accurately mapped the breakpoints of known and previously uncharacterized HBB deletions defining previously uncharacterized deletion breakpoints by PCR amplification and sequencing. The array also successfully identified the common HBA deletions --(SEA) and --(FIL). In summary, comparative genomic hybridization can be used to characterize deletions of the HBA and HBB loci, allowing high-resolution characterization of novel deletions that are not readily detected by PCR-based methods. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. 48 CFR 1845.7101-4 - Types of deletions from contractor property records.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Types of deletions from contractor property records. Contractors shall report the types of deletions... contract or other prime contract with the same contractor. (c) Transferred to NASA Center accountability. Deletion amounts that result from transfer of accountability to the NASA Center responsible for the...

  5. 48 CFR 1845.7101-4 - Types of deletions from contractor property records.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Types of deletions from contractor property records. Contractors shall report the types of deletions... contract or other prime contract with the same contractor. (c) Transferred to NASA Center accountability. Deletion amounts that result from transfer of accountability to the NASA Center responsible for the...

  6. 48 CFR 1845.7101-4 - Types of deletions from contractor property records.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Types of deletions from contractor property records. Contractors shall report the types of deletions... contract or other prime contract with the same contractor. (c) Transferred to NASA Center accountability. Deletion amounts that result from transfer of accountability to the NASA Center responsible for the...

  7. 48 CFR 1845.7101-4 - Types of deletions from contractor property records.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Types of deletions from contractor property records. Contractors shall report the types of deletions... contract or other prime contract with the same contractor. (c) Transferred to NASA Center accountability. Deletion amounts that result from transfer of accountability to the NASA Center responsible for the...

  8. 47 CFR 1.229 - Motions to enlarge, change, or delete issues.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 1 2013-10-01 2013-10-01 false Motions to enlarge, change, or delete issues. 1... Hearing Proceedings Participants and Issues § 1.229 Motions to enlarge, change, or delete issues. (a) A motion to enlarge, change or delete the issues may be filed by any party to a hearing. Except as provided...

  9. 17 CFR 145.4 - Public records available with identifying details deleted; nonpublic records available in...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... identifying details deleted; nonpublic records available in abridged or summary form. 145.4 Section 145.4... § 145.4 Public records available with identifying details deleted; nonpublic records available in... privacy, the Commission may delete identifying details when it makes available “public records” as defined...

  10. 17 CFR 145.4 - Public records available with identifying details deleted; nonpublic records available in...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... identifying details deleted; nonpublic records available in abridged or summary form. 145.4 Section 145.4... INFORMATION § 145.4 Public records available with identifying details deleted; nonpublic records available in... privacy, the Commission may delete identifying details when it makes available “public records” as defined...

  11. 47 CFR 1.229 - Motions to enlarge, change, or delete issues.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 1 2011-10-01 2011-10-01 false Motions to enlarge, change, or delete issues. 1... Hearing Proceedings Participants and Issues § 1.229 Motions to enlarge, change, or delete issues. Link to an amendment published at 76 FR 60672, September 29, 2011. (a) A motion to enlarge, change or delete...

  12. 41 CFR 51-6.8 - Deletion of items from the Procurement List.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... agency decides to request that the Committee delete a commodity or service from the Procurement List, it... the Government is no longer procuring the item in question, the Committee shall, prior to deleting an... of the decision to delete the item. Nonprofit agencies shall obtain concurrence of the contracting...

  13. Performance on Cloze Tests with Fixed-Ratio and Rational Deletions.

    ERIC Educational Resources Information Center

    Bachman, Lyle F.

    1985-01-01

    Describes a study that developed criteria for rationally selecting the words to delete in developing a cloze test. The study also tried to determine the extent to which performance on a cloze test with rational deletions differs from that on a cloze test developed by the fixed-ratio deletion procedure. (SED)

  14. 17 CFR 145.4 - Public records available with identifying details deleted; nonpublic records available in...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... identifying details deleted; nonpublic records available in abridged or summary form. 145.4 Section 145.4... § 145.4 Public records available with identifying details deleted; nonpublic records available in... privacy, the Commission may delete identifying details when it makes available “public records” as defined...

  15. 17 CFR 145.4 - Public records available with identifying details deleted; nonpublic records available in...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... identifying details deleted; nonpublic records available in abridged or summary form. 145.4 Section 145.4... § 145.4 Public records available with identifying details deleted; nonpublic records available in... privacy, the Commission may delete identifying details when it makes available “public records” as defined...

  16. 17 CFR 145.4 - Public records available with identifying details deleted; nonpublic records available in...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... identifying details deleted; nonpublic records available in abridged or summary form. 145.4 Section 145.4... § 145.4 Public records available with identifying details deleted; nonpublic records available in... privacy, the Commission may delete identifying details when it makes available “public records” as defined...

  17. 47 CFR 1.229 - Motions to enlarge, change, or delete issues.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 1 2014-10-01 2014-10-01 false Motions to enlarge, change, or delete issues. 1... Hearing Proceedings Participants and Issues § 1.229 Motions to enlarge, change, or delete issues. (a) A motion to enlarge, change or delete the issues may be filed by any party to a hearing. Except as provided...

  18. 41 CFR 51-6.8 - Deletion of items from the Procurement List.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... agency decides to request that the Committee delete a commodity or service from the Procurement List, it... the Government is no longer procuring the item in question, the Committee shall, prior to deleting an... of the decision to delete the item. Nonprofit agencies shall obtain concurrence of the contracting...

  19. Characterization of Deletions of the HBA and HBB Loci by Array Comparative Genomic Hybridization

    PubMed Central

    Sabath, Daniel E.; Bender, Michael A.; Sankaran, Vijay G.; Vamos, Esther; Kentsis, Alex; Yi, Hye-Son; Greisman, Harvey A.

    2017-01-01

    Thalassemia is among the most common genetic diseases worldwide. α-Thalassemia is usually caused by deletion of one or more of the duplicated HBA genes on chromosome 16. In contrast, most β-thalassemia results from point mutations that decrease or eliminate expression of the HBB gene on chromosome 11. Deletions within the HBB locus result in thalassemia or hereditary persistence of fetal Hb. Although routine diagnostic testing cannot distinguish thalassemia deletions from point mutations, deletional hereditary persistence of fetal Hb is notable for having an elevated HbF level with a normal mean corpuscular volume. A small number of deletions accounts for most α-thalassemias; in contrast, there are no predominant HBB deletions causing β-thalassemia. To facilitate the identification and characterization of deletions of the HBA and HBB globin loci, we performed array-based comparative genomic hybridization using a custom oligonucleotide microarray. We accurately mapped the breakpoints of known and previously uncharacterized HBB deletions defining previously uncharacterized deletion breakpoints by PCR amplification and sequencing. The array also successfully identified the common HBA deletions --SEA and --FIL. In summary, comparative genomic hybridization can be used to characterize deletions of the HBA and HBB loci, allowing high-resolution characterization of novel deletions that are not readily detected by PCR-based methods. PMID:26612711

  20. Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy

    PubMed Central

    Beedle, Aaron M.; Turner, Amy J.; Saito, Yoshiaki; Lueck, John D.; Foltz, Steven J.; Fortunato, Marisa J.; Nienaber, Patricia M.; Campbell, Kevin P.

    2012-01-01

    Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies. Here, we developed a dystroglycanopathy mouse model using inducible or muscle-specific promoters to conditionally disrupt fukutin (Fktn), a gene required for dystroglycan processing. In conditional Fktn-KO mice, we observed a near absence of functionally glycosylated dystroglycan within 18 days of gene deletion. Twenty-week-old KO mice showed clear dystrophic histopathology and a defect in glycosylation near the dystroglycan O-mannose phosphate, whether onset of Fktn excision driven by muscle-specific promoters occurred at E8 or E17. However, the earlier gene deletion resulted in more severe phenotypes, with a faster onset of damage and weakness, reduced weight and viability, and regenerating fibers of smaller size. The dependence of phenotype severity on the developmental timing of muscle Fktn deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional Fktn-KO mouse allows the generation of tissue- and timing-specific defects in dystroglycan glycosylation, avoids embryonic lethality, and produces a phenotype resembling patient pathology, it is a promising new model for the study of secondary dystroglycanopathy. PMID:22922256

  1. Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta.

    PubMed

    Poulter, James A; Murillo, Gina; Brookes, Steven J; Smith, Claire E L; Parry, David A; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

    2014-10-15

    Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid.

  2. Chlorambucil effectively induces deletion mutations in mouse germ cells.

    PubMed Central

    Russell, L B; Hunsicker, P R; Cacheiro, N L; Bangham, J W; Russell, W L; Shelby, M D

    1989-01-01

    The chemotherapeutic agent chlorambucil was found to be more effective than x-rays or any chemical investigated to date in inducing high yields of mouse germ-line mutations that appear to be deletions or other structural changes. Induction of mutations involving seven specific loci was studied after exposures of various male germ-cell stages to chlorambucil at 10-25 mg/kg. A total of 60,750 offspring was scored. Mutation rates in spermatogonial stem cells were not significantly increased over control values; this negative result is not attributable to selective elimination of mutant cells. Mutations were, however, clearly induced in treated post-stem-cell stages, among which marked variations in mutational response were found. Maximum yield occurred after exposure of early spermatids, with approximately 1% of all offspring carrying a specific-locus mutation in the 10 mg/kg group. The stage-response pattern for chlorambucil differs from that of all other chemicals investigated to date in the specific-locus test. Thus far, all but one of the tested mutations induced by chlorambucil in post-stem-cell stages have been proved deletions or other structural changes by genetic, cytogenetic, and/or molecular criteria. Deletion mutations have recently been useful for molecular mapping and for structure-function correlations of genomic regions. For generating presumed large-lesion germ-line mutations at highest frequencies, chlorambucil may be the mutagen of choice. Images PMID:2726748

  3. Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa.

    PubMed

    Le, Shuai; Yao, Xinyue; Lu, Shuguang; Tan, Yinling; Rao, Xiancai; Li, Ming; Jin, Xiaolin; Wang, Jing; Zhao, Yan; Wu, Nicholas C; Lux, Renate; He, Xuesong; Shi, Wenyuan; Hu, Fuquan

    2014-04-28

    Bacteria develop a broad range of phage resistance mechanisms, such as prevention of phage adsorption and CRISPR/Cas system, to survive phage predation. In this study, Pseudomonas aeruginosa PA1 strain was infected with lytic phage PaP1, and phage-resistant mutants were selected. A high percentage (~30%) of these mutants displayed red pigmentation phenotype (Red mutant). Through comparative genomic analysis, one Red mutant PA1r was found to have a 219.6 kb genomic fragment deletion, which contains two key genes hmgA and galU related to the observed phenotypes. Deletion of hmgA resulted in the accumulation of a red compound homogentisic acid; while A galU mutant is devoid of O-antigen, which is required for phage adsorption. Intriguingly, while the loss of galU conferred phage resistance, it significantly attenuated PA1r in a mouse infection experiment. Our study revealed a novel phage resistance mechanism via chromosomal DNA deletion in P. aeruginosa.

  4. Deletion of Prepl Causes Growth Impairment and Hypotonia in Mice

    PubMed Central

    Lone, Anna Mari; Leidl, Mathias; McFedries, Amanda K.; Horner, James W.; Creemers, John; Saghatelian, Alan

    2014-01-01

    Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL−/−) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL−/− mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL+/+) counterparts. A righting assay revealed that PREPL−/− pups took significantly longer than PREPL+/+ pups to right themselves when placed on their backs. This deficit indicates that PREPL−/− mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease. PMID:24586561

  5. Chlorambucil effectively induces deletion mutations in mouse germ cells

    SciTech Connect

    Russell, L.B.; Hunsicker, P.R.; Cacheiro, N.L.A.; Bangham, J.W.; Russell, W.L.; Shelby, M.D. )

    1989-05-01

    The chemotherapeutic agent chlorambucil was found to be more effective than x-rays or any chemical investigated to data in inducing high yields of mouse germ-line mutations that appear to be deletions or other structural changes. Induction of mutations involving seven specific loci was studied after exposures of various male germ-cell stages to chlorambucil at 10-25 mg/kg. A total of 60,750 offspring was scored. Mutation rates in spermatogonial stem cells were not significantly increased over control values; this negative result is not attributable to selective elimination of mutant cells. Mutations were, however, clearly induced in treated post-stem-cell stages, among which marked variations in mutational response were found. Maximum yield occurred after exposure of early spermatids, with {approx} 1% of all offspring carrying a specific-locus mutation in the 10 mg/kg group. The stage-response pattern for chlorambucil differs from that of all other chemicals investigated to date in the specific-locus test. Thus far, all but one of the tested mutations induced by chlorambucil in post-stem-cell stages have been proved deletions or other structural changes by genetic, cytogenetic, and/or molecular criteria. Deletion mutations have recently been useful for molecular mapping and for structure-function correlations of genomic regions. For generating presumed large-lesion germline mutations at highest frequencies, chlorambucil may be the mutagen of choice.

  6. Fatal dilated cardiomyopathy associated with a mitochondrial DNA deletion.

    PubMed

    Moslemi, A R; Selimovic, N; Bergh, C H; Oldfors, A

    2000-01-01

    A 27-year-old man was admitted to hospital because of severe cardiac failure. Investigation revealed dilated cardiomyopathy with a left ventricular ejection fraction of 15-20%. During adolescence the patient had been investigated for growth retardation and he also had progressive external ophthalmoplegia. There had been no symptoms of cardiac disease until 2 weeks before admittance. An endomyocardial biopsy showed cardiomyocytes deficient in cytochrome c oxidase (COX) in a mosaic pattern. A skeletal muscle biopsy showed mitochondrial myopathy with COX-deficient ragged-red fibers. Molecular genetic analysis revealed a heteroplasmic, 3.8-kb, mitochondrial DNA (mtDNA) deletion in heart and muscle. PCR-based quantification of the proportion of mtDNA with deletion showed 47% mutated mtDNA in the myocardial biopsy and 68% in muscle. In spite of treatment, the condition deteriorated and the patient died 5 days after admittance. This case demonstrates that mtDNA deletions may occasionally be the cause of severe dilated cardiomyopathy, and that morphological and molecular genetic diagnosis may be obtained by endomyocardial biopsy. Copyright 2000 S. Karger AG, Basel.

  7. Two new cases of FMRI deletion associated with mental impairment

    SciTech Connect

    Hirst, M.; Grewal, P.; Flannery, A.; Davies, K.; Slatter, R.; Maher, E.; Barton, D.; Fryns, J.P.

    1995-01-01

    Screening of families clinically ascertained for the fragile X syndrome phenotype revealed two mentally impaired males who were cytogenetically negative for the fragile X chromosome. In both cases, screening for the FMR1 trinucleotide expansion mutation revealed a rearrangement within the FMR1 gene. In the first case, a 660-bp deletion is present in 40% of peripheral lymphocytes. PCR and sequence analysis revealed it to include the CpG island and the CGG trinucleotide repeat, thus removing the FMR1 promoter region and putative mRNA start site. In the second case, PCR analysis demonstrated that a deletion extended from a point proximal to FMR1 to 25 kb into the gene, removing all the region 5{prime} to exon 11. The distal breakpoint was confirmed by Southern blot analysis and localized to a 600-bp region, and FMR1-mRNA analysis in a cell line established from this individual confirmed the lack of a transcript. These deletion patients provide further confirmatory evidence that loss of FMR1 gene expression is indeed responsible for mental retardation. Additionally, these cases highlight the need for the careful examination of the FMR1 gene, even in the absence of cytogenetic expression, particularly when several fragile X-like clinical features are present. 31 refs., 6 figs.

  8. Mitochondrial DNA exhibits resistance to induced point and deletion mutations

    PubMed Central

    Valente, William J.; Ericson, Nolan G.; Long, Alexandra S.; White, Paul A.; Marchetti, Francesco; Bielas, Jason H.

    2016-01-01

    The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed MutaTMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure. PMID:27550180

  9. Site deletion from the National Priorities List. CERCLA Information Brief

    SciTech Connect

    Whitehead, B.

    1993-11-01

    Section 105 of the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) of 1980, as amended by the Superfund Amendments and Reauthorization Act (SARA) of 1986, requires the US Environmental Protection Agency (EPA) to maintain a National Priorities List (NPL) of releases or potential releases of hazardous substances, pollutants, or contaminants that warrant further investigation to determine if they pose risks to human health and the environment. Typically a site is placed on the NPL based on its score derived by applying the Hazard Ranking System (HRS), a screening mechanism EPA uses to evaluate the relative threat to human health and the environment posed by the release, or potential release, of hazardous substances into the environment. Sites scoring 28.50 or greater are eligible for the NPL. Additionally, each state may designate one top-priority site, regardless of the HRS score. Infrequently, EPA may utilize provisions established under 40 CFR 300.425(c)(3) to place a site on the NPL. A site may be deleted from the NPL if it is determined that no further response is required to protect human health and the environment. To date, EPA has deleted 51 sites from the NPL. The criteria and procedures for deleting a site from the NPL, as established by the National Oil and Hazardous Substances Pollution Contingency Plan, otherwise known as the National Contingency Plan (NCP), and other relevant policies are the subject of this Information Brief.

  10. Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta

    PubMed Central

    Poulter, James A.; Murillo, Gina; Brookes, Steven J.; Smith, Claire E. L.; Parry, David A.; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F.; Mighell, Alan J.

    2014-01-01

    Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid. PMID:24858907

  11. Deletion mapping of the spinal muscular atrophy region

    SciTech Connect

    Burgein, L.; Lefebvre, S.; Buriet, P.

    1994-09-01

    Autosomal recessive childhood spinal muscular atrophies (SMA) are divided into severe (Type I) and mild forms (Types II and III). Using a combination of fine genetic and physical mapping, we constructed a YAC contig of the 5q13 region spanning the disease locus and showed the presence of low copy-repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in 9 unrelated SMA patients. Moreover, deletion events were statistically associated with type I SMA patients as shown by marked heterozygosity deficiency for the loci studied. Genetic analysis with all polymorphic DNA markers derived from the YAC contig has allowed to define the smallest rearrangement between the loci detected by C161 and the C212-C272-C171, covering a 1Mb interval. A large scale physical map of this region has been constructed using pulsed field gel electrophoresis (PFGE) and rare cutter restriction endonucleases. Comparison of the physical map has been established both in patients harboring deletions and unaffected controls using various anonymous probes derived from the YAC contig. This analysis has allowed us to define a physical interval in which at least a portion of the SMA gene must be located. Search for genes within this interval will contribute to the identification of the disease gene or genes.

  12. The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

    PubMed

    Zhu-Ge, Xiang-Kai; Pan, Zi-Hao; Tang, Fang; Mao, Xiang; Hu, Lin; Wang, Shao-Hui; Xu, Bin; Lu, Cheng-Ping; Fan, Hong-Jie; Dai, Jian-Jun

    2015-12-01

    Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.

  13. Physiological activation of Akt by PHLPP1 deletion protects against pathological hypertrophy.

    PubMed

    Moc, Courtney; Taylor, Amy E; Chesini, Gino P; Zambrano, Cristina M; Barlow, Melissa S; Zhang, Xiaoxue; Gustafsson, Åsa B; Purcell, Nicole H

    2015-02-01

    To examine the role of physiological Akt signalling in pathological hypertrophy through analysis of PHLPP1 (PH domain leucine-rich repeat protein phosphatase) knock-out (KO) mice. To investigate the in vivo requirement for 'physiological' control of Akt activation in cardiac growth, we examined the effect of deleting the Akt phosphatase, PHLPP, on the induction of cardiac hypertrophy. Basal Akt phosphorylation increased nearly two-fold in the cardiomyocytes from PHLPP1 KO mice and physiological hypertrophy induced by swimming exercise was accentuated as assessed by increased heart size and myocyte cell area. In contrast, the development of pathophysiological hypertrophy induced by pressure overload and assessed by increases in heart size, myocyte cell area, and hypertrophic gene expression was attenuated. This attenuation coincided with decreased fibrosis and cell death in the KO mice. Cast moulding revealed increased capillary density basally in the KO hearts, which was further elevated relative to wild-type mouse hearts in response to pressure overload. In vitro studies with isolated myocytes in co-culture also demonstrated that PHLPP1 deletion in cardiomyocytes can enhance endothelial tube formation. Expression of the pro-angiogenic factor VEGF was also elevated basally and accentuated in response to transverse aortic constriction in hearts from KO mice. Our data suggest that enhancing Akt activity by inhibiting its PHLPP1-mediated dephosphorylation promotes processes associated with physiological hypertrophy that may be beneficial in attenuating the development of pathological hypertrophy. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  14. A comprehensive analysis of the importance of translation initiation factors for Haloferax volcanii applying deletion and conditional depletion mutants.

    PubMed

    Gäbel, Katrin; Schmitt, Jessica; Schulz, Sebastian; Näther, Daniela J; Soppa, Jörg

    2013-01-01

    Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 - IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed.

  15. Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes.

    PubMed

    Pullen, A M; Marrack, P; Kappler, J W

    1989-05-01

    V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.

  16. Conditional deletion of Abca3 in alveolar type II cells alters surfactant homeostasis in newborn and adult mice

    PubMed Central

    Besnard, Valérie; Matsuzaki, Yohei; Clark, Jean; Xu, Yan; Wert, Susan E.; Ikegami, Machiko; Stahlman, Mildred T.; Weaver, Timothy E.; Hunt, Alan N.; Postle, Anthony D.

    2010-01-01

    ATP-binding cassette A3 (ABCA3) is a lipid transport protein required for synthesis and storage of pulmonary surfactant in type II cells in the alveoli. Abca3 was conditionally deleted in respiratory epithelial cells (Abca3Δ/Δ) in vivo. The majority of mice in which Abca3 was deleted in alveolar type II cells died shortly after birth from respiratory distress related to surfactant deficiency. Approximately 30% of the Abca3Δ/Δ mice survived after birth. Surviving Abca3Δ/Δ mice developed emphysema in the absence of significant pulmonary inflammation. Staining of lung tissue and mRNA isolated from alveolar type II cells demonstrated that ∼50% of alveolar type II cells lacked ABCA3. Phospholipid content and composition were altered in lung tissue, lamellar bodies, and bronchoalveolar lavage fluid from adult Abca3Δ/Δ mice. In adult Abca3Δ/Δ mice, cells lacking ABCA3 had decreased expression of mRNAs associated with lipid synthesis and transport. FOXA2 and CCAAT enhancer-binding protein-α, transcription factors known to regulate genes regulating lung lipid metabolism, were markedly decreased in cells lacking ABCA3. Deletion of Abca3 disrupted surfactant lipid synthesis in a cell-autonomous manner. Compensatory surfactant synthesis was initiated in ABCA3-sufficient type II cells, indicating that surfactant homeostasis is a highly regulated process that includes sensing and coregulation among alveolar type II cells. PMID:20190032

  17. Deletion of Plasmodium berghei-Specific CD4+ T Cells Adoptively Transferred into Recipient Mice after Challenge with Homologous Parasite

    NASA Astrophysics Data System (ADS)

    Hirunpetcharat, Chakrit; Good, Michael F.

    1998-02-01

    The immune response to malaria parasites includes T cell responses that reduce parasites by effector T cell responses and by providing help for antibody responses. Some parasites are more sensitive to antibody and others are more sensitive to cell-mediated immunity. We demonstrate that cultured CD4+ T cells that produce interferon CD4+ and interleukin 2, but not interleukin 4, in response to stimulation with the rodent parasite Plasmodium berghei can reduce but not eliminate parasites in vivo after adoptive transfer. Although cells can persist in vivo for up to 9 months in uninfected mice, infection results in elimination of up to 99% of specific T cells in different tissues, as judged by tracking T cells labeled with the fluorescent dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester. T cells specific for ovalbumin are unaffected. In vivo activation and division of transferred T cells per se are not responsible for deletion because T cells positive for 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester divide up to six times within 7 days in uninfected mice and are not deleted. Understanding the factors responsible for parasite-mediated specific deletion of T cells would enhance our knowledge of parasite immunity.

  18. Deletion variant in the ADRA2B gene increases coupling between emotional responses at encoding and later retrieval of emotional memories.

    PubMed

    Todd, R M; Müller, D J; Palombo, D J; Robertson, A; Eaton, T; Freeman, N; Levine, B; Anderson, A K

    2014-07-01

    A deletion variant of the ADRA2B gene that codes for the α2b adrenoceptor has been linked to greater susceptibility to traumatic memory as well as attentional biases in perceptual encoding of negatively valenced stimuli. The goal of the present study was to examine whether emotional enhancements of memory associated with the ADRA2B deletion variant were predicted by encoding, as indexed by the subjectively perceived emotional salience (i.e., arousal) of events at the time of encoding. Genotyping was performed on 186 healthy young adults who rated positive, negative, and neutral scenes for level of emotional arousal and subsequently performed a surprise recognition memory task 1 week later. Experience of childhood trauma was also measured, as well as additional genetic variations associated with emotional biases and episodic memory. Results showed that subjective arousal was linked to memory accuracy and confidence for ADRA2B deletion carriers but not for non-carriers. Our results suggest that carrying the ADRA2B deletion variant enhances the relationship between arousal at encoding and subsequent memory for moderately arousing events. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure.

    PubMed

    Arpino, James A J; Reddington, Samuel C; Halliwell, Lisa M; Rizkallah, Pierre J; Jones, D Dafydd

    2014-06-10

    Altering a protein's backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate residue deletion across the breadth of the protein, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFP(G4Δ)) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFP(G4Δ) retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFP(G4Δ) structure revealed that G4 deletion caused a beneficial helical registry shift resulting in a new polar interaction network, which potentially stabilizes a cis proline peptide bond and links secondary structure elements. Thus, deletion mutations and registry shifts can enhance proteins through structural rearrangements not possible by substitution mutations alone.

  20. Generating Bona Fide Mammalian Prions with Internal Deletions

    PubMed Central

    Munoz-Montesino, Carola; Sizun, Christina; Moudjou, Mohammed; Herzog, Laetitia; Reine, Fabienne; Chapuis, Jérôme; Ciric, Danica; Igel-Egalon, Angelique; Laude, Hubert; Béringue, Vincent; Rezaei, Human

    2016-01-01

    ABSTRACT Mammalian prions are PrP proteins with altered structures causing transmissible fatal neuro